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MALDI TIMS IMS Reveals Ganglioside Molecular Diversity within Murine S. aureus Kidney Tissue Abscesses
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  • Katerina V. Djambazova
    Katerina V. Djambazova
    Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee 37232, United States
    Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee 37232, United States
    More by Katerina V. Djambazova
    Orcidhttps://orcid.org/0000-0002-2680-9014
  • Katherine N. Gibson-Corley
    Katherine N. Gibson-Corley
    Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States
    More by Katherine N. Gibson-Corley
  • Jeffrey A. Freiberg
    Jeffrey A. Freiberg
    Vanderbilt Institute for Infection, Immunology and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States
    Division of Infectious Diseases, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States
    More by Jeffrey A. Freiberg
  • Richard M. Caprioli
    Richard M. Caprioli
    Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee 37232, United States
    Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States
    Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232, United States
    Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, United States
    Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232, United States
    Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, United States
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    Orcidhttps://orcid.org/0000-0001-5859-3310
  • Eric P. Skaar
    Eric P. Skaar
    Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States
    Vanderbilt Institute for Infection, Immunology and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States
    Vanderbilt Institute for Chemical Biology, Vanderbilt University, Nashville, Tennessee 37232, United States
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  • Jeffrey M. Spraggins*
    Jeffrey M. Spraggins
    Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee 37232, United States
    Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee 37232, United States
    Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States
    Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232, United States
    Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, United States
    *[email protected]
    More by Jeffrey M. Spraggins
    Orcidhttps://orcid.org/0000-0001-9198-5498
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Journal of the American Society for Mass Spectrometry

Cite this: J. Am. Soc. Mass Spectrom. 2024, 35, 8, 1692–1701
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https://doi.org/10.1021/jasms.4c00089
Published July 25, 2024

Copyright © 2024 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Gangliosides play important roles in innate and adaptive immunity. The high degree of structural heterogeneity results in significant variability in ganglioside expression patterns and greatly complicates linking structure and function. Structural characterization at the site of infection is essential in elucidating host ganglioside function in response to invading pathogens, such as Staphylococcus aureus (S. aureus). Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) enables high-specificity spatial investigation of intact gangliosides. Here, ganglioside structural and spatial heterogeneity within an S. aureus-infected mouse kidney abscess was characterized. Differences in spatial distributions were observed for gangliosides of different classes and those that differ in ceramide chain composition and oligosaccharide-bound sialic acid. Furthermore, integrating trapped ion mobility spectrometry (TIMS) allowed for the gas-phase separation and visualization of monosialylated ganglioside isomers that differ in sialic acid type and position. The isomers differ in spatial distributions within the host–pathogen interface, where molecular patterns revealed new molecular zones in the abscess previously unidentified by traditional histology.

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Introduction

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Staphylococcus aureus (S. aureus) is a widespread commensal Gram-positive bacterium and pathogen. (1−3) It is the leading cause of skin and soft tissue infections and can result in more serious complications, including bacteremia, pneumonia, and osteomyelitis. (4) Staphylococcal invasion typically leads to soft-tissue abscess formation, where the bacteria organize into staphylococcal abscess communities (SACs), surrounded by severe inflammation of neighboring tissues. (3−5) The mature abscess is a highly heterogeneous environment with a diverse subset of host- and pathogen-derived molecules. (5,6) Traditional histological approaches such as stained microscopy and immunohistochemistry (IHC) can reveal the abscess architecture and the spatial distributions of specific targets within the tissues. (3) However, detailed molecular information cannot be gleaned from these techniques, and more advanced analytical approaches, such as imaging mass spectrometry (IMS) are necessary to provide molecular and spatial context. (7)
Matrix-assisted laser desorption/ionization (MALDI) IMS allows for direct visualization of metabolites, lipids, peptides, and other biomolecules from tissue sections. (7−9) In a typical MALDI IMS experiment, tissue sections are thaw-mounted onto conductive glass slides and coated with a UV-absorbing matrix. A laser is rastered across the sample to produce individual mass spectra at each ablation position. Molecular images are generated by plotting the ion intensities as a heat map across all ablation spots (pixels). (10) MALDI IMS is a minimally destructive approach, which allows for downstream histology to be performed. (11,12) In this manner, histology and molecular information are correlated, allowing for a thorough investigation of the spatio-molecular characteristics of S. aureus infections. (13−17) With these multimodal approaches we can visualize and study molecules involved in host–pathogen interactions. (18−21)
Gangliosides (acidic glycosphingolipids) are immune system modulators that can alter both innate and adaptive immune functions. (20−22) They reside primarily in the cellular plasma membrane, where the ceramide anchors the molecule to the membrane and the glycan protrudes outward from the cell. (23) At the cell surface, gangliosides can interact with other molecules and regulate the activity of proteins. (19,20,24) Gangliosides can also be unintentional targets for microbial adhesion, where viruses, bacteria, and bacterial toxins bind to the carbohydrate of gangliosides on host cell surfaces. (18−20,22,25) Elucidating the distinct functions of gangliosides in response to invading pathogens is particularly challenging due to their vast structural diversity. Thus, comprehensive structural information on gangliosides at the site of infection is a critical first step in exploring ganglioside function in immune response and the interaction between host and pathogen.
Structurally, the ganglioside molecule can be divided into two functional units–a hydrophilic oligosaccharide headgroup carrying sialic acid(s) and a hydrophobic ceramide moiety. (Figure 1A). The ceramide moiety of the gangliosides is composed of a sphingosine and a fatty acyl chain (Figure 1B). The fatty acyls in gangliosides generally range from 16 to 30 carbons, with varying degrees of unsaturation. The most common sphingoid bases are d18:1 and d20:1, where 18 and 20 refer to the number of carbons in the chain, 1 indicates the presence of a single double bond, and d (“di” - two) denotes the number of hydroxyl groups on the sphingoid base. Other sphingoid bases present in eukaryotes include, but are not limited to, d18:0, d20:0, d16:0, and t18:1 (“tri” - three hydroxyl groups). Both hydrocarbon length and the number of double bonds of the ceramide can dictate biological function. The hydrophilic oligosaccharide headgroup is comprised of multiple saccharide units, including glucose (Glc), galactose (Gal), galactosamine (GalNAc), and sialic acids (Figure 1). (26) Gangliosides are synthesized by the stepwise addition of monosaccharides to the glucose-ceramide (GlcCer) unit (Figure S1). After the addition of Gal, the lactosylceramide (LacCer) serves as a building block for all subsequent gangliosides and is a branching point in the synthesis. From there, the oligosaccharide chain can be elongated by the addition of monosaccharides, creating the o-series gangliosides (GA2, GA1, GM1b). Alternatively, the sequential addition of sialic acids to LacCer by sialyltransferases form gangliosides GM3, GD3, GT3, etc. Each of these serves as a precursor for more complex gangliosides, belonging to the a-, b-, and c-series, respectively. The letter refers to the number of sialic acids bound to the internal galactose unit, resulting in o-(zero), a-(one), b-(two), c-(three) series gangliosides. (23,26−29) The differences in the oligosaccharide chain and the quantity/position of sialic acids greatly increase the number of possible structures and their biological roles.

Figure 1

Figure 1. Ganglioside molecular structure (GM3) example (A) highlights common diversities in the ceramide backbone (B) and common sialic acids and their alterations (C); Neu, neuraminic acid; KDN, deaminated neuraminic acid; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid.

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Finally, negatively charged sialic acids are essential components of the ganglioside molecule. They alone can influence ganglioside function, particularly in pathogen recognition and cell infiltration. (30,31) Although all sialic acids share the same 9-carbon carboxylated sugar, there are numerous structural differences and modifications that can be present. The most common ganglioside-bound sialic acids are N-acetylneuraminic acid (NeuAc) and N-glycolyneuraminic acid (NeuGc), (32) where common modifications include O-acetylation, N-acetylation, and N-glycolylation (Figure 1C). The diversity of sialic acid structures at various sialylation sites greatly increases the number of possible structures. For example, although ∼200 ganglioside structures have been described in the literature, considering the known diversity of the ceramide and the glycan headgroup, it is hypothesized that over 3000 unique gangliosides exist. (28,29)
Mass spectrometry has been at the forefront of ganglioside analysis, where liquid chromatography (LC)-based studies have provided detailed structural information. (33−35) MALDI IMS has been used to investigate the spatial distributions of these analytes (36−39) and gas-phase separations, such as with ion mobility, have provided further structural characterization in an imaging context, however, with limited specificity. (40,41) Recently, we demonstrated MALDI trapped ion mobility spectrometry (TIMS) IMS for the separation and visualization of a- and b-series GD1 isomers within rat brain and spinal cord tissues. (40−42) Briefly, TIMS separations are carried out in the first vacuum stage of a mass spectrometer in an augmented ion funnel composed of an entrance funnel, TIMS tunnel, and exit funnel. (43−45) Propelled by a carrier gas, ions are accumulated, trapped, and separated by an electric field. (46−48) To elute trapped ions, the electric field gradient is gradually reduced, releasing ions with ascending mobilities. TIMS IMS is capable of high resolving power separations (>200 in 50–500 ms), ideal for addressing structural heterogeneity and isomerism prevalent in this molecular class. (42,45,46,48,49)
Here, MALDI TIMS IMS of S. aureus-infected mouse kidney was used for in-depth structural characterization of gangliosides within an inflammatory lesion. The structural heterogeneity of gangliosides including ceramide composition, oligosaccharide chain, as well as sialic-acid positional information, was characterized. Furthermore, ganglioside isomers were resolved by TIMS separations, and their distinct distributions within the abscess were visualized.

Methods

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Materials

2′,5′-Dihydroxyacetophenone (DHA), hematoxylin, eosin, and ammonium sulfate were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade acetonitrile, and ethanol, were purchased from Fisher Scientific (Pittsburgh, PA, USA). Indium tin oxide-coated slides (CG-81IN-S115) were purchased from Delta Technologies, Limited (Loveland, CO, USA).

Murine Model of S. aureus Infection

Female C57BL/6J mice (6–8 weeks old) (Jackson Laboratory) were retro-orbitally infected with 1 × 107 colony forming units (CFU) of Staphylococcus aureus Newman in 100 μL of sterile phosphate-buffered saline as previously described. (13) Following infection, the mice were euthanized 10 days postinfection (DPI). The organs were removed, immediately frozen on a bed of dry ice, and stored at −80 °C until further processing. All animal experimental protocols were reviewed and approved by the Vanderbilt University Institutional Animal Care and Use Committee and were in compliance with institutional policies, NIH guidelines, the Animal Welfare Act, and American Veterinary Medical Association guidelines on euthanasia.

Sample Preparation

Infected (10 DPI) and control mouse kidney sections were cryosectioned to 10 μm thickness using a CM3050 S cryostat (Leica Biosystems, Wetzlar, Germany) and thaw-mounted onto conductive indium tin oxide-coated glass slides (Delta Technologies, Loveland, CO, USA). Matrix (2′,5′- DHA with 62.5 μM ammonium sulfate in 60% ethanol–water) was applied using a robotic sprayer (M5 Sprayer, HTX Technologies, NC, USA) for a final matrix density of 1.48 μg/mm2 (Table S1). Immediately following MALDI IMS, the matrix was removed from the sample using 100% ethanol and rehydrated with graded ethanol and H2O. Tissues were stained using an H&E stain. Brightfield microscopy was obtained at 20× magnification using a Zeis AxioScan Z1 slide scanner (Carl Zeiss Microscopy GmbH, Oberkochen, Germany).

MALDI TIMS IMS

All experiments were carried out on a prototype MALDI timsTOF fleX mass spectrometer (Bruker Daltonics, Bremen, Germany). (45) Data were acquired in negative ionization mode (m/z 1,000–3,000) at 50 μm (45 μm beam scan) spatial resolution with ∼50% laser power at 10 kHz, 400 shots per pixel, and 35,680 (infected) and 19,694 (control) pixels per sample. MALDI TIMS data were collected over a 1/K0 range of 1.50–2.45 V·s/cm2 (T3 Ramp: 230.5–124.7 V) with a ramp time of 450 ms, resulting in a scan rate (Sr) of 0.24 V/ms. The following parameters were kept constant across all imaging experiments: ESI dry gas temperature, 100 °C; Ion transfer time, 120 μs; prepulse storage time, 12 μs; collision RF, 3500 Vpp; TIMS funnel 1 RF, 450 Vpp; TIMS funnel 2 RF , 400 Vpp; multipole RF, 400 Vpp, collision cell entrance voltage, −200 V; MALDI deflection plate, 90 V. The source pressure was set to ∼2.35 mbar, to access higher 1/K0 ranges. Both MS and TIMS calibrations were performed using an Agilent ESI-L tuning mixture.

Identification and Data Analysis

Serial tissue sections were analyzed for further structural investigation. Fourier Transform-Ion Cyclotron Resonance (FT-ICR) MS (15T SolariX, Bruker Daltonics, Bremen, Germany) with ultrahigh-spectra resolution (∼200,000 at m/z 1544.86) provided exact mass measurements and eliminated the possibility of isobaric interferences. On-tissue MALDI TIMS MS/MS was collected from a small region of a serial tissue section for at least one representative species of each ganglioside class. Experimental details, such as sampled area (number of pixels), CID voltage, and isolation window width, are provided in the relevant supplemental figure captions. Table S2 summarizes all characteristic fragment ions used to make identification in this study. We retained ganglioside identifications if the mass error was <5 ppm by MALDI TIMS MS or FTICR MS; if the mass error was >5 ppm and no on-tissue fragmentation was collected for the species, data were removed from the final list of identified gangliosides. MALDI TIMS IMS data were analyzed and visualized using DataAnalysis and SCiLS (Bruker Daltonics, Bremen, Germany), respectively. All ion images were generated using a peak’s centroid m/z ± 10 mDa; images generated with both m/z and ion mobility information were generated with the following boundaries m/z ± 10 mDa and peak 1/K0 ± 0.007 V•s/cm2.

Results and Discussion

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Molecular and Structural Diversity within Murine Kidney Tissue Abscess

To investigate molecular heterogeneity at the host–pathogen interface, a 10 DPI mouse kidney tissue section was analyzed using MALDI TIMS IMS. Data were collected in negative ionization mode, where all analytes were detected as deprotonated [M-H]− ions. Average mass spectra comparing control and 10 DPI mouse kidney tissue sections can be seen in Figure S2, and the ion mobility heat map of the average mass spectrum of the 10 DPI mouse kidney data can be seen in Figure S3. An H&E stain of the infected sample was performed following IMS, and the microscopy image was histopathologically assessed to annotate known abscess morphology features by a board-certified Veterinary Pathologist (Figure 2A).

Figure 2

Figure 2. H&E Stain of 10 DPI S. aureus-infected mouse kidney section reveals several abscess lesions (blue boundaries) within the kidney section. A zoom-in of the lesion reveals abscess structures including a dark stained fibrous capsule (green), bacterial staphylococcal abscess communities (red arrows), a zone of healthy and dead immune cells (yellow arrow), and an intensely eosinophilic region, tentatively identified as the Splendore-Hoeppli phenomenon (bright blue dotted line) (A). Negative ion mode MALDI IMS images highlight different structures within the lesion (B).

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The SACs were identified at the center of the abscess (red arrows). The primary immune cells observed in the abscess were neutrophils, with degenerate neutrophils (pus) found near the bacterial colonies, and a mixture of necrotic and viable immune cells (most commonly neutrophils) found further from the abscess center (yellow arrow). The intensely eosinophilic material (bright blue boundary) is Splendore-Hoeppli phenomenon (reaction). (50−,52) This phenomenon is an in vivo formation of eosinophilic material, which has been observed around microorganisms like bacteria and fungi. While its’ nature and mechanism are not well understood, it is thought to be a deposition of antigen–antibody complexes and debris from the host inflammatory cells. (50,52) Finally, the microscopy also revealed a dark-stained fibrous capsule layer (fibrosis) at the outer abscess border (green), separating the abscess from the normal host tissue.
MALDI IMS data revealed a diverse molecular landscape within the abscess, as shown in Figure 2B. The infected mouse was sacrificed during late-stage infection (10 DPI), therefore the bacteria occupy a relatively small area of the lesion, as the majority of the abscess is made up of infiltrating immune cells and cellular debris. Bacterial cardiolipin CL (64:0) (m/z 1351.98, pink) revealed the spatial distributions of the bacteria within the abscess, and GM3 NeuAc (d34:1) (m/z 1151.71, red) colocalized with the fibrous capsule and the glomeruli. (15,53,54) While the distributions of these ions correlate well with known anatomical features, other ions reveal molecular heterogeneity not evident in the microscopy. For example, sulfated hexosylceramide SHexCer (d40:6) (m/z 1005.51, yellow) and GM2 NeuGc (d34:1) (m/z 1370.78, green) reveal distinct layers within the zone of healthy/necrotic immune cells, where the histology revealed a largely homogeneous layer. GM2 NeuGc (d34:1) also colocalized to the intensely eosinophilic region peripheral to the bacteria, identified as the Splendore-Hoeppli phenomenon. This reaction has been previously observed by histological assessment and is thought to be made up of glycoproteins, lipids, and calcium derived from host leukocytes. (50,51) However, the molecular makeup of Splendore-Hoeppli remains unknown; approaches like MALDI IMS offer a unique insight into the molecular landscape of this unique morphology.

Ganglioside Heterogeneity within Murine S. aureus Soft Tissue Abscesses

MALDI TIMS IMS was performed in negative ionization mode; all gangliosides were detected as deprotonated [M-H]− ions. Six different classes of gangliosides, including monosialylated (GM1, GM2, and GM3) and disialylated (GD1) were detected within the S. aureus-infected mouse kidney section (Figures 3, S2, and S3). Rare GalNAc-GM1b and extended series-GM1b gangliosides were also observed. These species have only been observed in a few immune cell types, including T-cells, B-cells, and Macrophages; however, their functions and abundance in other immune cells are not well understood. (21,55)

Figure 3

Figure 3. MALDI TIMS IMS shows structural and spatial diversity within a 10 DPI S.aureus-infected mouse kidney section across ganglioside classes: GM3 (A), GM2 (B), GM1 (C), GD1 (D), GalNAc-GM1b (E), and extended GM1b (F). No mobility information was used to generate the ion images.

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GM3 species are the simplest a-series gangliosides, composed of two carbohydrate units and a single sialic acid. A total of 11 GM3 species were detected in the infected kidney, where the ceramide chains ranged from C34 to C42 in length (Table S3, Figure 3A). Monounsaturated dihydroxylated ceramide chains were most common, and no unsaturated species were detected. In terms of sialic acid diversity, both NeuAc and NeuGc sialic acids were detected. GM3s were the only ganglioside species observed in both the control and infected tissue; a total of 8 GM3 species were observed in the control (Table S3, Figure S4). For example, m/z 1151.71, identified as GM3 NeuAc(d34:1), localized to the fibrous capsule of the abscess and the glomeruli (kidney filtering units). All GM3s localized to the fibrous capsule and were not detected in the intra-abscess region.
GM2s are synthesized from GM3s with the addition of GalNAc to the oligosaccharide chain to form a trisaccharide headgroup with a single sialic acid residue. A total of four GM2 species were detected in the infected tissue sample (Figure 3B, Table S4, Figure S5) All GM2s had a dihydroxylated ceramide backbone, and only NeuGc sialic acids were detected. An example on-tissue MALDI MS/MS spectrum is shown for GM2 NeuGC (d34:1) (m/z 1370.78) in Figure S6. All ions localized to the outer abscess layers, and signals were also observed neighboring the center of the abscess (Splendore-Hoeppli phenomenon). While the exact role of GM2s within infection processes is not completely understood, (21) it has been noted that GM2s can serve as receptors for bacterial toxin binding. (18)
The most abundant ganglioside class detected in the infected mouse kidney sample was GM1. GM1s contain a tetra-saccharide core and a single sialic acid. GM1s have high structural diversity resulting in several possible isomeric structures. In this example, 17 different GM1s were differentiated by mass alone, and an additional 8 were revealed by TIMS analysis (Figure 3C, Tables S5 and S7). The ceramide chain compositions ranged from C34 to C42, where fully unsaturated, di- and tri-unsaturated ceramide chains were observed. Dihydroxylated ceramides were most common, however, several gangliosides with trihydroxylated chains were also detected. GM1s were detected throughout various intra-abscess structures and in the surrounding immune cell infiltrates.
GD1s are disialylated gangliosides, comprised of a tetrasaccharide carbohydrate chain with two sialic acids. Numerous GD1 species, as highlighted in Table S6 and Figure S8, were detected in the S. aureus-infected mouse kidney (Figure 3D). Polysialylated species, such as GD1, can be particularly challenging to analyze, due to the in-source fragmentation of the labile sialic acid bond, as well as the high number of possible isomeric structures. Therefore, confident identification of all GD1s could not be achieved. More complex extended o-series gangliosides, such as GalNAc-GM1b, and extended series GM1b species are synthesized from GM1b gangliosides by the sequential addition of GalNAc, and Gal-GalNAc, respectively. Here, 16 different GalNAc-GM1b and three extended series GM1b gangliosides were detected (Table S7, Figure S9). Structures were confirmed with on-tissue MS/MS, where an example fragmentation spectrum for GalNAc-GM1b (d42:1) is highlighted in Figure S10. The spatial distributions of these gangliosides can be seen in Figure 3E and F. While Sarbu et al. (56) recently described GalNAc-GD1 isomers in cerebrospinal fluid, to our knowledge, GalNAc- and extended GM1b gangliosides have not been detected or visualized with MALDI IMS to date. Little is known about the biological function of extended series gangliosides. They have been reported as part of T-cell membranes, where they have been linked to T-cell differentiation and activation. (20,21,24) However, our results indicate that they may also be crucial components of other immune cells since infection would primarily activate innate immune cells like neutrophils and macrophages. More extended series- and GalNAc-GM1b gangliosides are likely present in the data, however, close isobaric overlap with GD1 species hinders confident identification with MALDI TIMS MS/MS. It is important to note that polysialylated gangliosides are prone to in-source fragmentation. Loss of sialic acid(s) can be observed, resulting in artificially high intensities of monosialylated species and misidentification of in-source fragments as endogenous molecules. While MALDI IMS is a soft ionization technique that can minimize this effect, in-source fragmentation may still be observed. Further optimization of instrument parameters and sample preparation strategies can enhance MALDI IMS of gangliosides.

Ganglioside Isomers

Monosialylated GM1s can have a single sialic acid on the internal galactose unit (GM1a) or on the terminal galactose unit (GM1b, o-series). Sialic acid diversity is also prevalent, where both NeuAc and NeuGc are common in mammalian non-neuronal cells. Here, MALDI TIMS IMS was used to separate, visualize, and identify GM1 isomers that differ by type and position of the sialic acid. Conformational changes can occur in the gas phase when ions are subjected to prolonged storage and separation times; however, based on previous work by our group, we do not expect conformational changes to play a significant role in GM1 isomer separations, as presented here. (42,49) All detected GM1 isomers are listed in Tables S8 and S9.

GM1a and GM1b Isomers

All GM1 a- and o-series isomers identified by MALDI TIMS IMS are listed in Table S8. Two examples are highlighted in Figure 4. Here, the extracted ion mobilograms of m/z 1516.84 (GM1 NeuAc (d34:1)) and m/z 1626.95 (GM1 NeuAc (d42:2)) revealed two resolved ions (Figure 4A and B). To illustrate the unique spatial distributions of each isomeric species, both the m/z and 1/K0 information were used to generate the ion images. Each panel highlights the distinct spatial distributions of the lower mobility ion in red, the higher mobility ion in blue, and an overlay of both ion images. In both examples, the more compact structures localized to the internal regions of the abscess, whereas the higher mobility ions localized to the external boundary of the abscess and to regions of immune cell infiltration beyond the defined abscesses. Subsequent on-tissue MALDI TIMS MS/MS collected from a serial tissue section was used to identify the isomers. Example fragmentation data is shown for m/z 1626.95 (Figure S11). Here, the fragmentation confirmed the ganglioside class (GM1), the type of sialic acid present (NeuAc), the ceramide chain composition (d42:2), and the presence of both GM1b and GM1a isomers (via their preferential/unique fragments). In particular, the fragment ions at m/z 1217.83 (Cer-Glc-Gal-NeuAC) and m/z 1173.81 (Cer-Glc-Gal-GalNAc) allowed for the differentiation of a- and o-series isomers, respectively. The extracted ion mobilograms of the fragments were used to link each isomer to its corresponding mobility of the parent ion. It was concluded that the lower mobility ion was GM1b (red), and the higher - GM1a (blue).

Figure 4

Figure 4. Extracted ion mobilograms of m/z 1516.84 (A) and m/z 1626.94 (B) reveal the TIMS separation of GM1b (red) and GM1a (blue) in S.aureus-infected mouse kidney section. Ion images of GM1b (red), GM1a (blue), and an overlay of both can be seen for GM1(d34:1) and GM1(d42:2) in (A) and (B), respectively.

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NeuAc-tCer and NeuGc-dCer Ganglioside Isomers

NeuAc-tCer and NeuGc-dCer GM1 isomers arise when a NeuGc-containing GM1 with a dihydroxylated ceramide chain overlaps in mass with a NeuAc sialic acid with a trihydroxylated ceramide chain. Table S9 summarizes the possible NeuGc-dCer and NeuAc-tCer isomers within the data set. Figure 5 highlights GM1 NeuGc (d34:1) and GM1 NeuAc (t34:1), detected at m/z 1532.83. Additionally, both gangliosides can be GM1a and GM1b, resulting in at least four possible isomers under a single m/z (Figure S12). The extracted ion mobilogram of m/z 1532.83 reveals several gas-phase conformers (Figure 5A): 1/K0 1.90 (red), 1/K0 1.93 (pink), 1/K0 1.94 (blue), 1/K0 1.97 (gray), and 1/K0 2.01 (light blue). Each of the m/z + 1/K0 ion distributions can be seen in the ion images in Figure 5B. While all ions localize to the lesions and areas of immune cell infiltration, subtle differences in their spatial distributions can be seen. For example, GM1a NeuGC (d34:1) (green) localizes to the ray-like structure beyond the lower left abscess, denoted with a solid arrow. This isomer is also more intense around the abscess borders and near primary zones of immune cell infiltration. Both GM1b NeuAC (d34:1) (pink) and GM1a NeuGC (d34:1) (light blue) have higher intensities in the outer boundary of the lesion (dotted arrow), where the GM1a NeuAc (t34:1) (blue) is not as prevalent. Finally, the unidentified isomer (white) cannot be seen in the abscesses, but is found throughout the outer cortex of the kidney, possibly indicating areas of immune cell infiltrates, not yet organized into the abscesses.

Figure 5

Figure 5. Extracted ion mobilogram of m/z 1532.83 reveals five partially resolved peaks: 1/K0 1.90 (red), 1/K0 1.93 (pink), 1/K0 1.94 (blue), 1/K0 1.97 (gray/white), and 1/K0 2.01 (light blue) (A), where each m/z + 1/K0 ion distribution can be seen in the ion images (B). Ions were identified with on-tissue MALDI TIMS MS/MS, as seen in Figure 6.

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On-tissue MALDI TIMS MS/MS was performed on a serial tissue section to identify the conformers. The fragmentation spectrum revealed the presence of both GM1 NeuAc (t34:1) and GM1 NeuGc (d34:1), shown in Figure 6A and 6B, respectively. The sialic acid structures were confirmed by the neutral loss of NeuAc (- m/z 291.13) and NeuGc (- m/z 307.09). The ceramide compositions Cer(t34:1) and Cer(d34:1) were confirmed by fragments at m/z 536.49 and m/z 553.51, respectively (Figure 6A and B). To assign identities of the parent-peak resolved conformers, the extracted ion mobilograms of fragment ions were generated where applicable. The extracted ion mobilograms of m/z 1225.74 and m/z 860.56, indicate that GM1 NeuGc (d34:1) is associated with peaks 1/K0 1.93(1) (pink) and 1/K0 2.01 (light blue). The presence of two peaks in the extracted fragment ion mobilogram also indicates the presence of both GM1a and GM1b NeuAc (t34:1) isomers. Similarly, the extracted ion mobilograms of m/z 1241.70 and m/z 876.56, revealed that the peaks at 1/K0 1.91 (red) and 1/K0 1.93(2) (blue) arise from GM1 NeuAc (t34:1) – both GM1a and GM1b isomers. Lastly, the extracted ion mobilogram of m/z 876.56 revealed two additional isomers, likely arising from ceramide chain diversity, that could not be identified (Figure 6B). To confirm the presence of a- and o-series isomers, unique (or preferential) fragments of each series and their extracted ion mobilograms were studied (Figure 6C and D). The fragment at m/z 1167.71 is unique for GM1a (both NeuGc (d34:1) and NeuAC (t34:1)). This fragment arises from the loss of Gal-GalNAc from the headgroup, while the sialic acid remains intact on the internal Gal unit. (57) The extracted ion mobilogram also revealed that NeuAc (t34:1) is the more intense ion. The preferential fragmentation of GM1b gangliosides includes a stepwise loss of a sialic acid, and a Gal unit. (34,57) For GM1 NeuAc (t34:1) and GM1 NeuGc (d34:1), this results in fragment ions m/z 1079.67, and m/z 1063.69, respectively. From the extracted ion mobilogram of each, two peaks can be seen. The lower mobility peaks are attributed to the o-series isomers (GM1b), however, higher-mobility a-series fragmentation was also observed. A more comprehensively annotated fragmentation spectrum is included in the Supporting Information (Figure S13).

Figure 6

Figure 6. On-tissue MALDI TIMS MS/MS of m/z 1532.83 reveals the presence of isomeric GM1 Neu5Gc (d34:1) (A) and GM1 Neu5Ac (t34:1) (B) in a 10 DPI S.aureus-infected mouse kidney. For both GM1 Neu5Gc (d34:1) and GM1 Neu5Ac (t34:1), GM1a and GM1b isomers were also identified, as evidenced by (C) and (D), respectively.

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Ultimately, we confirmed the presence of four different isomers - NeuAc (t34:1) and NeuGc (d34:1), both a- and o-series of each, and mapped their distinct spatial distributions (Figure 5) within the infection lesion. These findings indicate that the major isomer in the mixture is GM1a NeuAC (t34:1), 1/K0 2.01; therefore, the molecular image of m/z 1532.83 would be dominated by this isomer without mobility separation.

Conclusions

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The work described herein investigates ganglioside structural and spatial heterogeneity within a soft tissue S. aureus abscess. MALDI TIMS IMS revealed the spatial distributions of gangliosides that varied by class, ceramide chain composition, and sialic acid type and position. While some ions localized to known abscess architecture, others revealed molecular heterogeneity, which could not be discerned by pathohistological assessment. Integrating TIMS allowed for gas-phase separation of GM1b, GM1a, NeuAc-tCer, and NeuGc-dCer isomers within the abscess. This level of structural specificity is essential in elucidating the distinct functions isomeric gangliosides have at the site of infection. For example, previous studies have implicated GM1b isomers and NeuGc-containing gangliosides in host–pathogen interactions; and researchers have postulated that ganglioside patterns of immune cells vary significantly throughout the course of an infection. (55,58,59) Although significant strides have been made toward understanding the role of gangliosides within infections, much of the knowledge remains descriptive. (18) Comprehensive analyses, as described herein, are integral to pushing this research beyond characterization. Gaining fundamental insight into the metabolism of gangliosides, and glycosphingolipids as a whole, (18) is a promising avenue of research for potential treatment and/or prevention of infectious diseases.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jasms.4c00089.

  • Ganglioside synthesis pathway; average mass spectrum of control and infected mouse kidney section; ion mobility heat map of 10 DPI mouse kidney average mass spectrum; table summarizing matrix deposition parameters; table summarizing characteristic fragment ions used for ganglioside identification; tables and figures of gangliosides identified in a 10 DPI mouse kidney section, including GM3, GM2, GM1, GalNAc- and extended series GM1b, and GD1; on-tissue fragmentation of GM2 and GalNAc-GM1b; tables listing ganglioside isomers identified in 10 DPI mouse kidney, including GM1a and GM1b, and NeuAc-tCer and NeuGc-dCer; on-tissue fragmentation of a- and o-series ganglioside isomers (m/z 1626.95); molecular structures of GM1 isomers detected at m/z 1532.83; detailed on-tissue fragmentation of m/z 1532.83 (PDF)

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Author Information

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  • Corresponding Author
    • Jeffrey M. Spraggins - Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee 37232, United States;  Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee 37232, United States;  Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States;  Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232, United States;  Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, United States;  Orcidhttps://orcid.org/0000-0001-9198-5498;  Email: [email protected]
  • Authors
    • Katerina V. Djambazova - Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee 37232, United States;  Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee 37232, United States;  Orcidhttps://orcid.org/0000-0002-2680-9014
    • Katherine N. Gibson-Corley - Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States
    • Jeffrey A. Freiberg - Vanderbilt Institute for Infection, Immunology and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States;  Division of Infectious Diseases, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States
    • Richard M. Caprioli - Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee 37232, United States;  Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States;  Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232, United States;  Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, United States;  Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232, United States;  Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, United States;  Orcidhttps://orcid.org/0000-0001-5859-3310
    • Eric P. Skaar - Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States;  Vanderbilt Institute for Infection, Immunology and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States;  Vanderbilt Institute for Chemical Biology, Vanderbilt University, Nashville, Tennessee 37232, United States
  • Author Contributions

    The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We would like to acknowledge Dr. Andy Weiss for his efforts on the mouse infection model. This work was funded by grant support provided by the National Institute of Health (NIH) including grants U01DK133766 (awarded to J.M.S.), U54DK134302 (awarded to J.M.S.), R01AG078803 (awarded to J.M.S.), U54EY032442 (awarded to J.M.S.), R01AI145992 (awarded to J.M.S. and E.P.S.), R01AI138581 (awarded to J.M.S. and E.P.S), and R01AI150701 (awarded to E.P.S.). K.V.D. was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) training grant (T32DK007569-34). J.A.F. was supported by the National Institute of Allergy and Infectious Diseases (NIAID) training grant (T32AI007540) and a postdoctoral fellowship (F32AI169905).

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    Nakayama, H.; Nagafuku, M.; Suzuki, A.; Iwabuchi, K.; Inokuchi, J.-I. The Regulatory Roles of Glycosphingolipid-Enriched Lipid Rafts in Immune Systems. FEBS Lett. 2018, 592, 3921– 3942,  DOI: 10.1002/1873-3468.13275
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    Zhang, T.; Waard, A. A. D.; Wuhrer, M.; Spaapen, R. M. The Role of Glycosphingolipids in Immune Cell Functions. Frontiers in Immunology 2019, 10, 90,  DOI: 10.3389/fimmu.2019.00090
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    Potapenko, M.; Shurin, G. V.; de León, J. Gangliosides As Immunomodulators. Advances in Experimental Medicine and Biology 2007, 601, 195– 203,  DOI: 10.1007/978-0-387-72005-0_20
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    Muggli, T.; Bühr, C.; Schürch, S. Challenges in the Analysis of Gangliosides by LC-MS. CHIMIA 2022, 76, 109,  DOI: 10.2533/chimia.2022.109
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    Inokuchi, J. I.; Nagafuku, M.; Ohno, I.; Suzuki, A. Heterogeneity of Gangliosides among T Cell Subsets. Cell. Mol. Life Sci. 2013, 70, 3067– 3075,  DOI: 10.1007/s00018-012-1208-x
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    Takahashi, T.; Suzuki, T. Role of Sulfatide in Normal and Pathological Cells and Tissues. J. Lipid Res. 2012, 53, 1437– 1450,  DOI: 10.1194/jlr.R026682
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    Role of sulfatide in normal and pathological cells and tissues
    Takahashi, Tadanobu; Suzuki, Takashi
    Journal of Lipid Research (2012), 53 (8), 1437-1450CODEN: JLPRAW; ISSN:0022-2275. (American Society for Biochemistry and Molecular Biology, Inc.)
    A review. Sulfatide is 3-O-sulfogalactosylceramide that is synthesized by two transferases (ceramide galactosyltransferase and cerebroside sulfotransferase) from ceramide and is specifically degraded by a sulfatase (arylsulfatase A). Sulfatide is a multifunctional mol. for various biol. fields including the nervous system, insulin secretion, immune system, hemostasis/thrombosis, bacterial infection, and virus infection. Therefore, abnormal metab. or expression change of sulfatide could cause various diseases. Here, we discuss the important biol. roles of sulfatide in the nervous system, insulin secretion, immune system, hemostasis/thrombosis, cancer, and microbial infections including human immunodeficiency virus and influenza A virus. Our review will be helpful to achieve a comprehensive understanding of sulfatide, which serves as a fundamental target of prevention of and therapy for nervous disorders, diabetes mellitus, immunol. diseases, cancer, and infectious diseases.
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    Yu, R. K.; Tsai, Y. T.; Ariga, T.; Yanagisawa, M. Structures, Biosynthesis, and Functions of Gangliosides-an Overview. J. Oleo Sci. 2011, 60, 537– 544,  DOI: 10.5650/jos.60.537
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    Structures, biosynthesis, and functions of gangliosides-an overview
    Yu, Robert K.; Tsai, Yi-Tzang; Ariga, Toshio; Yanagisawa, Makoto
    Journal of Oleo Science (2011), 60 (10), 537-544CODEN: JOSOAP; ISSN:1345-8957. (Japan Oil ChemistsÏ Society)
    A review. Gangliosides are sialic acid-contg. glycosphingolipids that are most abundant in the nervous system. Heterogeneity and diversity of the structures in their carbohydrate chains are characteristic hallmarks of these lipids; so far, 188 gangliosides with different carbohydrate structures have been identified in vertebrates. The mol. structural complexity increases manifold if one considers heterogeneity in the lipophilic components. The expression levels and patterns of brain gangliosides are known to change drastically during development. In cells, gangliosides are primarily, but not exclusively, localized in the outer leaflets of plasma membranes and are integral components of cell surface microdomains with sphingomyelin and cholesterol from which they participate in cell-cell recognition, adhesion, and signal transduction. In this brief review, we discuss the structures, metab. and functions of gangliosides.
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    AWASTHI, Y. C.; SRIVASTAVA, S. K. STRUCTURE, FUNCTION AND METABOLISM OF GLYCOSPHINGOLIPIDS. Biochemistry of Brain 1980, 1– 20,  DOI: 10.1016/B978-0-08-021345-3.50004-6
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    Lunghi, G.; Fazzari, M.; Biase, E. D.; Mauri, L.; Chiricozzi, E.; Sonnino, S. The Structure of Gangliosides Hides a Code for Determining Neuronal Functions. FEBS Open Biol. 2021, 11, 3193,  DOI: 10.1002/2211-5463.13197
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    Novaconi, C. R.; Onulov, R.; Serb, A. F.; Sisu, E.; Dinca, N.; Pascariu, M.-C.; Georgescu, M. Assessing Glycosphingolipid Profiles in Human Health and Disease Using Non-Imaging MALDI Mass Spectrometry. Applied Sciences 2023, Vol. 13, Page 9922 2023, 13, 9922,  DOI: 10.3390/app13179922
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    Diversity in the sialic acids
    Varki, Ajit
    Glycobiology (1992), 2 (1), 25-40CODEN: GLYCE3; ISSN:0959-6658.
    A review, with many refs., of the occurrence, structure, biochem., and biol. significance of the diversity of sialic acids in oligosaccharides and sialoglycoproteins. Sialic acid nomenclature is also discussed.
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    Ghosh, S. Sialic Acid and Biology of Life: An Introduction. Sialic Acids and Sialoglycoconjugates in the Biology of Life, Health and Disease 2020, 1,  DOI: 10.1016/B978-0-12-816126-5.00001-9
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    Ito, E.; Tominaga, A.; Waki, H.; Miseki, K.; Tomioka, A.; Nakajima, K.; Kakehi, K.; Suzuki, M.; Taniguchi, N.; Suzuki, A. Structural Characterization of Monosialo-, Disialo- and Trisialo-Gangliosides by Negative Ion AP-MALDI-QIT-TOF Mass Spectrometry with MSn Switching. Neurochem. Res. 2012, 37, 1315– 1324,  DOI: 10.1007/s11064-012-0735-z
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    Barrientos, R. C.; Zhang, Q. Recent Advances in the Mass Spectrometric Analysis of Glycosphingolipidome - A Review. Anal. Chim. Acta 2020, 1132, 134– 155,  DOI: 10.1016/j.aca.2020.05.051
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    Recent advances in the mass spectrometric analysis of glycosphingolipidome - A review
    Barrientos, Rodell C.; Zhang, Qibin
    Analytica Chimica Acta (2020), 1132 (), 134-155CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
    A review. Aberrant expression of glycosphingolipids has been implicated in a myriad of diseases, but the authors' understanding of the strucural diversity, spatial distribution, and biol. function of this class of biomols. remains limited. These challenges partly stem from a lack of sensitive tools that can detect, identify, and quantify glycosphingolipids at the mol. level. Mass spectrometry has emerged as a powerful tool poised to address most of these challenges. Here, the authors review the recent developments in anal. glycosphingolipidomics with an emphasis on sample prepn., mass spectrometry and tandem mass spectrometry-based structural characterization, label-free and labeling-based quantification. The authors also discuss the nomenclature of glycosphingolipids, and emerging technologies like ion mobility spectrometry in differentiation of glycosphingolipid isomers. The intrinsic advantages and shortcomings of each method are carefully critiqued in line with an individual's research goals. Finally, future perspectives on anal. sphingolipidomics are stated, including a need for novel and more sensive methods in isomer sepn., low abundance species detection, and profiling the spatial distribution of glycosphingolipid mol. species in cells and tissues using imaging mass spectrometry.
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    Li, H.; Liu, Y.; Wang, Z.; Xie, Y.; Yang, L.; Zhao, Y.; Tian, R. Mass Spectrometry-Based Ganglioside Profiling Provides Potential Insights into Alzheimer’s Disease Development. Journal of Chromatography A 2022, 1676, 463196,  DOI: 10.1016/j.chroma.2022.463196
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    Suteanu-Simulescu, A.; Sarbu, M.; Ica, R.; Petrica, L.; Zamfir, A. D. Ganglioside Analysis in Body Fluids by Liquid-Phase Separation Techniques Hyphenated to Mass Spectrometry. Electrophoresis 2023, 44, 501– 520,  DOI: 10.1002/elps.202200229
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    Yang, E.; Dufresne, M.; Chaurand, P. Enhancing Ganglioside Species Detection for MALDI-TOF Imaging Mass Spectrometry in Negative Reflectron Mode. Int. J. Mass Spectrom. 2019, 437, 3– 9,  DOI: 10.1016/j.ijms.2017.09.011
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    Enhancing ganglioside species detection for MALDI-TOF imaging mass spectrometry in negative reflectron mode
    Yang, Ethan; Dufresne, Martin; Chaurand, Pierre
    International Journal of Mass Spectrometry (2019), 437 (), 3-9CODEN: IMSPF8; ISSN:1387-3806. (Elsevier B.V.)
    Enhanced ganglioside species detection was achieved for matrix assisted laser desorption ionization time-of-flight imaging mass spectrometry (MALDI-TOF IMS) in neg. reflectron mode using a novel sample prepn. protocol that involves washing the tissue in ammonium salt solns. followed by spray depositing ammonium salts and waiting 24 h after sublimation of 1,5-diaminonaphthalene (DAN) before data acquisition. Application of this novel method to normal adult mouse brains led to more than 10-fold increase in total ion intensity in the ganglioside mass range and an increase in the no. of detected sialylated species from 3 to 15, with no apparent delocalization obsd. at 20μm spatial resoln., making it a powerful technique with the potential to provide greater information about gangliosides in numerous biol. contexts.
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    Colsch, B.; Jackson, S. N.; Dutta, S.; Woods, A. S. Molecular Microscopy of Brain Gangliosides: Illustrating Their Distribution in Hippocampal Cell Layers. ACS Chem. Neurosci. 2011, 2, 213– 222,  DOI: 10.1021/cn100096h
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    Molecular Microscopy of Brain Gangliosides: Illustrating their Distribution in Hippocampal Cell Layers
    Colsch, Benoit; Jackson, Shelley N.; Dutta, Sucharita; Woods, Amina S.
    ACS Chemical Neuroscience (2011), 2 (4), 213-222CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)
    Gangliosides are amphiphilic mols. found in the outer layer of plasma membranes of all vertebrate cells. They play a major role in cell recognition and signaling and are involved in diseases affecting the central nervous system (CNS). We are reporting the differential distribution of ganglioside species in the rat brain's cerebrum, based on their ceramide assocd. core, and for the first time the presence of acetylation detected by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, which was used to map and image gangliosides with detailed structural information and histol. accuracy. In the hippocampus, localization of the major species GM1, GD1, O-acetylGD1, GT1, and O-acetylGT1 depends on the sphingoid base (d18:1 sphingosine or d20:1 eicosasphingosine) in the mol. layer of the dentate gyrus (ML), which is made up of three distinct layers, the inner mol. layer (IML), which contains sphingosine exclusively, and the middle mol. layer (MML) and the outer mol. layer (OML) where eicosasphingosine is the only sphingoid base. These results demonstrate that there is a different distribution of gangliosides in neuronal axons and dendrites depending on the ceramide core of each layer. GM3, GM2, GD3, and GD2 contain sphingosine predominantly and are mainly present in body cell layers, which are made up of the pyramidal cell layer (Py) and the granular layer of the dentate gyrus (GL), in contrast with GQ1 and the O-acetylated forms of GD1, GT1, and GQ1 gangliosides, which contain both sphingoid bases. However their distribution is based on the sialylated and acetylated oligosaccharide chains in the neuronal cell bodies.
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    Harris, A.; Roseborough, A.; Mor, R.; Yeung, K. K.-C.; Whitehead, S. N. Ganglioside Detection from Formalin-Fixed Human Brain Tissue Utilizing MALDI Imaging Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2020, 31, 479– 487,  DOI: 10.1021/jasms.9b00110
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    Ganglioside Detection from Formalin-Fixed Human Brain Tissue Utilizing MALDI Imaging Mass Spectrometry
    Harris, Aaron; Roseborough, A.; Mor, Rahul; Yeung, Ken K.-C.; Whitehead, Shawn N.
    Journal of the American Society for Mass Spectrometry (2020), 31 (3), 479-487CODEN: JAMSEF; ISSN:1879-1123. (American Chemical Society)
    Matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is used to perform mass spectrometric anal. directly on biol. samples providing visual and anatomical spatial information on mols. within tissues. A current obscuration of MALDI-IMS is that it is largely performed on fresh frozen tissue, whereas clin. tissue samples stored long-term are fixed in formalin, and the fixation process is thought to cause signal suppression for lipid mols. Studies have shown that fresh frozen tissue sections applied with an ammonium formate (AF) wash prior to matrix application in the MALDI-IMS procedure display an increase in obsd. signal intensity and sensitivity for lipid mols. detected within the brain while maintaining the spatial distribution of mols. throughout the tissue. In this work, we investigate the viability of formalin-fixed tissue imaging in a clin. setting by comparing MALDI data of fresh frozen and postfixed rat brain samples, along with postfixed human brain samples washed with AF to assess the capabilities of ganglioside anal. in MALDI imaging of formalin-fixed tissue. Results herein demonstrate that MALDI-IMS spectra for gangliosides, including GM1, were significantly enhanced in fresh frozen rat brain, formalin-fixed rat brain, and formalin-fixed human brain samples through the use of an AF wash. Improvements in MALDI-IMS image quality were demonstrated, and the spatial distribution of mols. was retained. Results indicate that this method will allow for the anal. of gangliosides from formalin-fixed clin. samples, which can open addnl. avenues for neurodegenerative disease research.
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    Zhang, Y.; Wang, J.; Liu, J.; Han, J.; Xiong, S.; Yong, W.; Zhao, Z. Combination of ESI and MALDI Mass Spectrometry for Qualitative, Semi-Quantitative and in Situ Analysis of Gangliosides in Brain. Sci. Rep. 2016, 6, 1– 11,  DOI: 10.1038/srep25289
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    Multivariate time series analysis on the dynamic relationship between Class B notifiable diseases and gross domestic product (GDP) in China
    Zhang, Tao; Yin, Fei; Zhou, Ting; Zhang, Xing-Yu; Li, Xiao-Song
    Scientific Reports (2016), 6 (1), 1-10CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
    The surveillance of infectious diseases is of great importance for disease control and prevention, and more attention should be paid to the Class B notifiable diseases in China. Meanwhile, according to the International Monetary Fund (IMF), the annual growth of Chinese gross domestic product (GDP) would decelerate below 7% after many years of soaring. Under such circumstances, this study aimed to answer what will happen to the incidence rates of infectious diseases in China if Chinese GDP growth remained below 7% in the next five years. Firstly, time plots and cross-correlation matrixes were presented to illustrate the characteristics of data. Then, the multivariate time series (MTS) models were proposed to explore the dynamic relationship between incidence rates and GDP. Three kinds of MTS models, i.e., vector auto-regressive (VAR) model for original series, VAR model for differenced series and error-correction model (ECM), were considered in this study. The rank of error-correction term was taken as an indicator for model selection. Finally, our results suggested that four kinds of infectious diseases (epidemic hemorrhagic fever, pertussis, scarlet fever and syphilis) might need attention in China because their incidence rates have increased since the year 2010.
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    Škrášková, K.; Claude, E.; Jones, E. A.; Towers, M.; Ellis, S. R.; Heeren, R. M. A. Enhanced Capabilities for Imaging Gangliosides in Murine Brain with Matrix-Assisted Laser Desorption/Ionization and Desorption Electrospray Ionization Mass Spectrometry Coupled to Ion Mobility Separation. Methods 2016, 104, 69– 78,  DOI: 10.1016/j.ymeth.2016.02.014
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    Enhanced capabilities for imaging gangliosides in murine brain with matrix-assisted laser desorption/ionization and desorption electrospray ionization mass spectrometry coupled to ion mobility separation
    Skraskova, Karolina; Claude, Emmanuelle; Jones, Emrys A.; Towers, Mark; Ellis, Shane R.; Heeren, Ron M. A.
    Methods (Amsterdam, Netherlands) (2016), 104 (), 69-78CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)
    The increased interest in lipidomics calls for improved yet simplified methods of lipid anal. Over the past two decades, mass spectrometry imaging (MSI) has been established as a powerful technique for the anal. of mol. distribution of a variety of compds. across tissue surfaces. Matrix-assisted laser desorption/ionization (MALDI) MSI is widely used to study the spatial distribution of common lipids. However, a thorough sample prepn. and necessity of vacuum for efficient ionization might hamper its use for high-throughput lipid anal. Desorption electrospray ionization (DESI) is a relatively young MS technique. In DESI, ionization of mols. occurs under ambient conditions, which alleviates sample prepn. Moreover, DESI does not require the application of an external matrix, making the detection of low mass species more feasible due to the lack of chem. matrix background. However, irresp. of the ionization method, the final information obtained during an MSI expt. is very complex and its anal. becomes challenging. It was shown that coupling MSI to ion mobility sepn. (IMS) simplifies imaging data interpretation. Here we employed DESI and MALDI MSI for a lipidomic anal. of the murine brain using the same IMS-enabled instrument. We report for the first time on the DESI IMS-MSI of multiply sialylated ganglioside species, as well as their acetylated versions, which we detected directly from the murine brain tissue. We show that poly-sialylated gangliosides can be imaged as multiply charged ions using DESI, while they are clearly sepd. from the rest of the lipid classes based on their charge state using ion mobility. This represents a major improvement in MSI of intact fragile lipid species. We addnl. show that complementary lipid information is reached under particular conditions when DESI is compared to MALDI MSI.
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    Jackson, S. N.; Colsch, B.; Egan, T.; Lewis, E. K.; Schultz, J. A.; Woods, A. S. Gangliosides’ Analysis by MALDI-Ion Mobility MS. Analyst 2011, 136, 463– 466,  DOI: 10.1039/C0AN00732C
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    Gangliosides' analysis by MALDI-ion mobility MS
    Jackson, Shelley N.; Colsch, Benoit; Egan, Thomas; Lewis, Ernest K.; Schultz, J. Albert; Woods, Amina S.
    Analyst (Cambridge, United Kingdom) (2011), 136 (3), 463-466CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
    The combination of ion mobility with matrix-assisted laser desorption/ionization allows for the rapid sepn. and anal. of biomols. in complex mixts. (such as tissue sections and cellular exts.), as isobaric lipid, peptide, and oligonucleotide mol. ions are pre-sepd. in the mobility cell before mass anal. MALDI-IM MS was used to analyze gangliosides, a class of complex glycosphingolipids that has different degrees of sialylation. Both GD1a and GD1b, structural isomers, were studied to see the effects on gas-phase structure depending upon the localization of the sialic acids. A total ganglioside ext. from mouse brain was also analyzed to measure the effectiveness of ion mobility to sep. out the different ganglioside species in a complex mixt.
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    Djambazova, K. V.; Dufresne, M.; Migas, L. G.; Kruse, A. R. S.; Plas, R. V. de; Caprioli, R. M.; Spraggins, J. M. MALDI TIMS IMS of Disialoganglioside Isomers─GD1a and GD1b in Murine Brain Tissue. Anal. Chem. 2023, 95, 1176,  DOI: 10.1021/acs.analchem.2c03939
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    MALDI TIMS IMS of Disialoganglioside Isomers-GD1a and GD1b in Murine Brain Tissue
    Djambazova, Katerina V.; Dufresne, Martin; Migas, Lukasz G.; Kruse, Angela R. S.; Van de Plas, Raf; Caprioli, Richard M.; Spraggins, Jeffrey M.
    Analytical Chemistry (Washington, DC, United States) (2023), 95 (2), 1176-1183CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Gangliosides are acidic glycosphingolipids, contg. ceramide moieties and oligosaccharide chains with one or more sialic acid residue(s) and are highly diverse isomeric structures with distinct biol. roles. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) enables the untargeted spatial anal. of gangliosides, among other biomols., directly from tissue sections. Integrating trapped ion mobility spectrometry with MALDI IMS allows for the anal. of isomeric lipid structures in situ. Here, we demonstrate the gas-phase sepn. and identification of disialoganglioside isomers GD1a and GD1b that differ in the position of a sialic acid residue, in multiple samples, including a std. mixt. of both isomers, a biol. ext., and directly from thin tissue sections. The unique spatial distributions of GD1a/b (d36:1) and GD1a/b (d38:1) isomers were detd. in rat hippocampus and spinal cord tissue sections, demonstrating the ability to structurally characterize and spatially map gangliosides based on both the carbohydrate chain and ceramide moieties.
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    Fernandez-Lima, F. A.; Kaplan, D. A.; Park, M. A. Note: Integration of Trapped Ion Mobility Spectrometry with Mass Spectrometry. Rev. Sci. Instrum. 2011, 82, 126106,  DOI: 10.1063/1.3665933
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    Note: Integration of trapped ion mobility spectrometry with mass spectrometry
    Fernandez-Lima, F. A.; Kaplan, D. A.; Park, M. A.
    Review of Scientific Instruments (2011), 82 (12), 126106/1-126106/3CODEN: RSINAK; ISSN:0034-6748. (American Institute of Physics)
    The integration of a trapped ion mobility spectrometer (TIMS) with a mass spectrometer (MS) for complementary fast, gas-phase mobility sepn. prior to mass anal. (TIMS-MS) is described. The ion transmission and mobility sepn. are discussed as a function of the ion source condition, bath gas velocity, anal. scan speed, RF ion confinement, and downstream ion optical conditions. TIMS mobility resoln. depends on the anal. scan speed and the bath gas velocity, with the unique advantage that the IMS sepn. can be easily tuned from high speed (≈25 ms) for rapid anal. to slower scans for higher mobility resoln. (R >80). (c) 2011 American Institute of Physics.
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    Fernandez-Lima, F.; Kaplan, D. A.; Suetering, J.; Park, M. A. Gas-Phase Separation Using a Trapped Ion Mobility Spectrometer. International journal for ion mobility spectrometry 2011, 14, 93,  DOI: 10.1007/s12127-011-0067-8
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    Spraggins, J. M.; Djambazova, K. V.; Rivera, E. S.; Migas, L. G.; Neumann, E. K.; Fuetterer, A.; Suetering, J.; Goedecke, N.; Ly, A.; Plas, R. V. D.; Caprioli, R. M. High-Performance Molecular Imaging with MALDI Trapped Ion-Mobility Time-of-Flight (TimsTOF) Mass Spectrometry. Anal. Chem. 2019, 91, 14552– 14560,  DOI: 10.1021/acs.analchem.9b03612
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    High-Performance Molecular Imaging with MALDI Trapped Ion-Mobility Time-of-Flight (timsTOF) Mass Spectrometry
    Spraggins, Jeffrey M.; Djambazova, Katerina V.; Rivera, Emilio S.; Migas, Lukasz G.; Neumann, Elizabeth K.; Fuetterer, Arne; Suetering, Juergen; Goedecke, Niels; Ly, Alice; Van de Plas, Raf; Caprioli, Richard M.
    Analytical Chemistry (Washington, DC, United States) (2019), 91 (22), 14552-14560CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Imaging mass spectrometry (IMS) enables the spatially targeted mol. assessment of biol. tissues at cellular resolns. New developments and technologies are essential for uncovering the mol. drivers of native physiol. function and disease. Instrumentation must maximize spatial resoln., throughput, sensitivity, and specificity, because tissue imaging expts. consist of thousands to millions of pixels. Here, the authors report the development and application of a matrix-assisted laser desorption/ionization (MALDI) trapped ion mobility spectrometry imaging platform. This prototype MALDI timsTOF instrument is capable of 10 μm spatial resolns. and 20 pixels/s throughput mol. imaging. The MALDI source utilizes a Bruker SmartBeam 3-D laser system that can generate a square burn pattern of <10 × 10 μm at the sample surface. General image performance was assessed using murine kidney and brain tissues and demonstrate that high spatial resoln. imaging data can be generated rapidly with mass measurement errors < 5 ppm and ∼40,000 resolving power. Initial TIMS-based imaging expts. were performed on whole body mouse pup tissue demonstrating the sepn. of closely isobaric [PC(32:0)+Na]+ and [PC(34:3)+H]+ (3 mDa mass difference) in the gas-phase. The authors have shown that the MALDI timsTOF platform can maintain reasonable data acquisition rates (>2 pixels/s) while providing the specificity necessary to differentiate components in complex mixts. of lipid adducts. The combination of high spatial resoln. and throughput imaging capabilities with high-performance TIMS sepns. provides a uniquely tunable platform to address many challenges assocd. with advanced mol. imaging applications.
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    Michelmann, K.; Silveira, J. A.; Ridgeway, M. E.; Park, M. A. Fundamentals of Trapped Ion Mobility Spectrometry. J. Am. Soc. Mass Spectrom. 2015, 26, 14– 24,  DOI: 10.1007/s13361-014-0999-4
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    Fundamentals of Trapped Ion Mobility Spectrometry
    Michelmann, Karsten; Silveira, Joshua A.; Ridgeway, Mark E.; Park, Melvin A.
    Journal of the American Society for Mass Spectrometry (2015), 26 (1), 14-24CODEN: JAMSEF; ISSN:1044-0305. (Springer)
    Trapped ion mobility spectrometry (TIMS) is a relatively new gas-phase sepn. method that was coupled to quadrupole orthogonal acceleration time-of-flight mass spectrometry. The TIMS analyzer is a segmented radiofrequency ion guide wherein ions are mobility-analyzed using an elec. field that holds ions stationary against a moving gas, unlike conventional drift tube ion mobility spectrometry where the gas is stationary. Ions are initially trapped, and subsequently eluted from the TIMS analyzer over time according to their mobility (K). Though TIMS has achieved a high level of performance (R > 250) in a small device (<5 cm) using modest operating potentials (<300 V), a proper theory has yet to be produced. Here, the authors develop a quant. theory for TIMS via math. derivation and simulations. A 1-dimensional anal. model, used to predict the transit time and theor. resolving power, is described. Theor. trends are in agreement with exptl. measurements performed as a function of K, pressure, and the axial elec. field scan rate. The linear dependence of the transit time with 1/K provides a fundamental basis for detn. of reduced mobility or collision cross section values by calibration. The quant. description of TIMS provides an operational understanding of the analyzer, outlines the current performance capabilities, and provides insight into future avenues for improvement. [Figure not available: see fulltext.].
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    Silveira, J. A.; Michelmann, K.; Ridgeway, M. E.; Park, M. A. Fundamentals of Trapped Ion Mobility Spectrometry Part II: Fluid Dynamics. J. Am. Soc. Mass Spectrom. 2016, 27, 585– 595,  DOI: 10.1007/s13361-015-1310-z
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    Fundamentals of Trapped Ion Mobility Spectrometry Part II: Fluid Dynamics
    Silveira, Joshua A.; Michelmann, Karsten; Ridgeway, Mark E.; Park, Melvin A.
    Journal of the American Society for Mass Spectrometry (2016), 27 (4), 585-595CODEN: JAMSEF; ISSN:1044-0305. (Springer)
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  1. Erin H. Seeley. Maximizing Data Coverage through Eight Sequential Mass Spectrometry Images of a Single Tissue Section. Journal of the American Society for Mass Spectrometry 2025, Article ASAP.
  2. Robert A. Shepherd, Conrad A. Fihn, Alex J. Tabag, Shaun M. K. McKinnie, Laura M. Sanchez. ‘Need for speed: high throughput’ – mass spectrometry approaches for high-throughput directed evolution screening of natural product enzymes. Natural Product Reports 2025, 20 https://doi.org/10.1039/D4NP00053F
  3. Christopher J. Good, Casey E. Butrico, Madeline E. Colley, Lauren N. Emmerson, Katherine N. Gibson-Corley, James E. Cassat, Jeffrey M. Spraggins, Richard M. Caprioli. Uncovering lipid dynamics in Staphylococcus aureus osteomyelitis using multimodal imaging mass spectrometry. Cell Chemical Biology 2024, 31 (10) , 1852-1868.e5. https://doi.org/10.1016/j.chembiol.2024.09.005
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  • Abstract

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    Figure 1

    Figure 1. Ganglioside molecular structure (GM3) example (A) highlights common diversities in the ceramide backbone (B) and common sialic acids and their alterations (C); Neu, neuraminic acid; KDN, deaminated neuraminic acid; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid.

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    Figure 2

    Figure 2. H&E Stain of 10 DPI S. aureus-infected mouse kidney section reveals several abscess lesions (blue boundaries) within the kidney section. A zoom-in of the lesion reveals abscess structures including a dark stained fibrous capsule (green), bacterial staphylococcal abscess communities (red arrows), a zone of healthy and dead immune cells (yellow arrow), and an intensely eosinophilic region, tentatively identified as the Splendore-Hoeppli phenomenon (bright blue dotted line) (A). Negative ion mode MALDI IMS images highlight different structures within the lesion (B).

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    Figure 3

    Figure 3. MALDI TIMS IMS shows structural and spatial diversity within a 10 DPI S.aureus-infected mouse kidney section across ganglioside classes: GM3 (A), GM2 (B), GM1 (C), GD1 (D), GalNAc-GM1b (E), and extended GM1b (F). No mobility information was used to generate the ion images.

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    Figure 4

    Figure 4. Extracted ion mobilograms of m/z 1516.84 (A) and m/z 1626.94 (B) reveal the TIMS separation of GM1b (red) and GM1a (blue) in S.aureus-infected mouse kidney section. Ion images of GM1b (red), GM1a (blue), and an overlay of both can be seen for GM1(d34:1) and GM1(d42:2) in (A) and (B), respectively.

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    Figure 5

    Figure 5. Extracted ion mobilogram of m/z 1532.83 reveals five partially resolved peaks: 1/K0 1.90 (red), 1/K0 1.93 (pink), 1/K0 1.94 (blue), 1/K0 1.97 (gray/white), and 1/K0 2.01 (light blue) (A), where each m/z + 1/K0 ion distribution can be seen in the ion images (B). Ions were identified with on-tissue MALDI TIMS MS/MS, as seen in Figure 6.

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    Figure 6

    Figure 6. On-tissue MALDI TIMS MS/MS of m/z 1532.83 reveals the presence of isomeric GM1 Neu5Gc (d34:1) (A) and GM1 Neu5Ac (t34:1) (B) in a 10 DPI S.aureus-infected mouse kidney. For both GM1 Neu5Gc (d34:1) and GM1 Neu5Ac (t34:1), GM1a and GM1b isomers were also identified, as evidenced by (C) and (D), respectively.

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      Buchberger, Amanda Rae; DeLaney, Kellen; Johnson, Jillian; Li, Lingjun
      Analytical Chemistry (Washington, DC, United States) (2018), 90 (1), 240-265CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      A review. Mass spectrometry imaging (MSI) is a powerful tool that enables untargeted studies into the spatial distribution of mol. species in a variety of samples. It has the capability to image thousands of mols., such as metabolites, lipids, peptides, proteins, and glycans, in a single expt. without labeling. The combination of information gained from mass spectrometry (MS) and visualization of spatial distributions in thin sample sections makes this a valuable chem. anal. tool useful for biol. specimen characterization. After minimal but careful sample prepn., the general setup of an MSI expt. involves defining an (x, y) grid over the surface of the sample, with the grid area chosen by the user. The mass spectrometer then ionizes the mols. on the surface of the sample and collects a mass spectrum at each pixel on the section, with the resulting spatial resoln. defined by the pixel size. After collecting the spectra, computational software can be used to select an individual mass-to-charge (m/z) value, and the intensity of the m/z is extd. from each pixel's spectrum. These intensities are then combined into a heat map image depicting the relative distribution of that m/z value throughout the sample's surface. In order to det. the identity of a specific m/z value, tandem MS (MS/MS) fragmentation can be performed on ions from each pixel, and the fragments can be used to piece together the structure of the unknown mol. Otherwise, the mol. can be identified based on its intact mass by accurate mass matching to databases of known mols. within a certain mass error range. Overall, the aim of this review is to provide an informative resource for those in the MSI community who are interested in improving MSI data quality and anal. or using MSI for novel applications. Particularly, advances from the last two years in sample prepn., instrumentation, quantitation, statistics, and multi-modal imaging that have allowed MSI to emerge as a powerful technique in various biomedical applications including clin. settings are discussed. Also, several novel biol. applications are highlighted to demonstrate the potential for the future of the MSI field.
    10. 10
      El-Aneed, A.; Cohen, A.; Banoub, J. Mass Spectrometry, Review of the Basics: Electrospray, MALDI, and Commonly Used Mass Analyzers. Appl. Spectrosc. Rev. 2009, 44, 210– 230,  DOI: 10.1080/05704920902717872
      10
      Mass Spectrometry, Review of the Basics: Electrospray, MALDI, and Commonly Used Mass Analyzers
      El-Aneed, Anas; Cohen, Aljandro; Banoub, Joseph
      Applied Spectroscopy Reviews (2009), 44 (3), 210-230CODEN: APSRBB; ISSN:0570-4928. (Taylor & Francis, Inc.)
      A review. Mass spectrometry (MS) has progressed to become a powerful anal. tool for both quant. and qual. applications. The first mass spectrometer was constructed in 1912 and since then it has developed from only analyzing small inorg. mols. to biol. macromols., practically with no mass limitations. Proteomics research, in particular, increasingly depends on MS technologies. The ability of mass spectrometry analyzing proteins and other biol. exts. is due to the advances gained through the development of soft ionization techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) that can transform biomols. into ions. ESI can efficiently be interfaced with sepn. techniques enhancing its role in the life and health sciences. MALDI, however, has the advantage of producing singly charges ions of peptides and proteins, minimizing spectral complexity. Regardless of the ionization source, the sensitivity of a mass spectrometer is related to the mass analyzer where ion sepn. occurs. Both quadrupole and time of flight (ToF) mass analyzers are commonly used and they can be configured together as QToF tandem mass spectrometric instruments. Tandem mass spectrometry (MS/MS), as the name indicates, is the result of performing two or more sequential sepns. of ions usually coupling two or more mass analyzers. Coupling a quadrupole and time of flight resulted in the prodn. of high-resoln. mass spectrometers (i.e., Q-ToF). This article will historically introduce mass spectrometry and summarizes the advantages and disadvantages of ESI and MALDI along with quadrupole and ToF mass analyzers, including the tech. marriage between the two analyzers. This article is educational in nature and intended for graduate students and senior biochem. students as well as chemists and biochemists who are not familiar with mass spectrometry and would like to learn the basics; it is not intended for mass spectrometry experts.
    11. 11
      Walch, A.; Rauser, S.; Deininger, S. O.; Höfler, H. MALDI Imaging Mass Spectrometry for Direct Tissue Analysis: A New Frontier for Molecular Histology. Histochemistry and Cell Biology 2008, 130, 421– 434,  DOI: 10.1007/s00418-008-0469-9
      11
      MALDI imaging mass spectrometry for direct tissue analysis: a new frontier for molecular histology
      Walch, Axel; Rauser, Sandra; Deininger, Soeren-Oliver; Hoefler, Heinz
      Histochemistry and Cell Biology (2008), 130 (3), 421-434CODEN: HCBIFP; ISSN:0948-6143. (Springer)
      A review. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating the distribution of proteins and small mols. within biol. systems through the in situ anal. of tissue sections. MALDI-IMS can det. the distribution of hundreds of unknown compds. in a single measurement and enables the acquisition of cellular expression profiles while maintaining the cellular and mol. integrity. In recent years, a great many advances in the practice of imaging mass spectrometry have taken place, making the technique more sensitive, robust, and ultimately useful. In this review, the authors focus on the current state of the art of MALDI-IMS, describe basic technol. developments for MALDI-IMS of animal and human tissues, and discuss some recent applications in basic research and in clin. settings.
    12. 12
      Chung, H.; Huang, P.; Chen, C.; Lee, C.; Hsu, C. Next-generation Pathology Practices with Mass Spectrometry Imaging. Mass Spectrom. Rev. 2023, 42, 2446  DOI: 10.1002/mas.21795
      There is no corresponding record for this reference.
    13. 13
      Corbin, B. D.; Seeley, E. H.; Raab, A.; Feldmann, J.; Miller, M. R.; Torres, V. J.; Anderson, K. L.; Dattilo, B. M.; Dunman, P. M.; Gerads, R.; Caprioli, R. M.; Nacken, W.; Chazin, W. J.; Skaar, E. P. Metal Chelation and Inhibition of Bacterial Growth in Tissue Abscesses. Science 2008, 319, 962– 965,  DOI: 10.1126/science.1152449
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      Metal Chelation and Inhibition of Bacterial Growth in Tissue Abscesses
      Corbin, Brian D.; Seeley, Erin H.; Raab, Andrea; Feldmann, Joerg; Miller, Michael R.; Torres, Victor J.; Anderson, Kelsi L.; Dattilo, Brian M.; Dunman, Paul M.; Gerads, Russell; Caprioli, Richard M.; Nacken, Wolfgang; Chazin, Walter J.; Skaar, Eric P.
      Science (Washington, DC, United States) (2008), 319 (5865), 962-965CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Bacterial infection often results in the formation of tissue abscesses, which represent the primary site of interaction between invading bacteria and the innate immune system. The authors identify the host protein calprotectin as a neutrophil-dependent factor expressed inside Staphylococcus aureus abscesses. Neutrophil-derived calprotectin inhibited S. aureus growth through chelation of nutrient Mn2+ and Zn2+: an activity that results in reprogramming of the bacterial transcriptome. The abscesses of mice lacking calprotectin were enriched in metal, and staphylococcal proliferation was enhanced in these metal-rich abscesses. These results demonstrate that calprotectin is a crit. factor in the innate immune response to infection and define metal chelation as a strategy for inhibiting microbial growth inside abscessed tissue.
    14. 14
      Cassat, J. E.; Moore, J. L.; Wilson, K. J.; Stark, Z.; Prentice, B. M.; Plas, R. V. de; Perry, W. J.; Zhang, Y.; Virostko, J.; Colvin, D. C.; Rose, K. L.; Judd, A. M.; Reyzer, M. L.; Spraggins, J. M.; Grunenwald, C. M.; Gore, J. C.; Caprioli, R. M.; Skaar, E. P. Integrated Molecular Imaging Reveals Tissue Heterogeneity Driving Host-Pathogen Interactions. Science Translational Medicine 2018, 10, eaan6361,  DOI: 10.1126/scitranslmed.aan6361
      There is no corresponding record for this reference.
    15. 15
      Perry, W. J.; Grunenwald, C. M.; Plas, R. V. de; Witten, J. C.; Martin, D. R.; Apte, S. S.; Cassat, J. E.; Pettersson, G. B.; Caprioli, R. M.; Skaar, E. P.; Spraggins, J. M. Visualizing Staphylococcus Aureus Pathogenic Membrane Modification within the Host Infection Environment by Multimodal Imaging Mass Spectrometry. Cell Chemical Biology 2022, 29, 1209– 1217,  DOI: 10.1016/j.chembiol.2022.05.004
      There is no corresponding record for this reference.
    16. 16
      Angeletti, S.; Ciccozzi, M. Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry in Clinical Microbiology: An Updating Review. Infection, Genetics and Evolution 2019, 76, 104063,  DOI: 10.1016/j.meegid.2019.104063
      There is no corresponding record for this reference.
    17. 17
      Moore, J. L.; Caprioli, R. M.; Skaar, E. P. Advanced Mass Spectrometry Technologies for the Study of Microbial Pathogenesis. Curr. Opin. Microbiol. 2014, 19, 45– 51,  DOI: 10.1016/j.mib.2014.05.023
      17
      Advanced mass spectrometry technologies for the study of microbial pathogenesis
      Moore, Jessica L.; Caprioli, Richard M.; Skaar, Eric P.
      Current Opinion in Microbiology (2014), 19 (), 45-51CODEN: COMIF7; ISSN:1369-5274. (Elsevier Ltd.)
      A review. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been successfully applied to the field of microbial pathogenesis with promising results, principally in diagnostic microbiol. to rapidly identify bacteria based on the mol. profiles of small cell populations. Direct profiling of mols. from serum and tissue samples by MALDI MS provides a means to study the pathogen-host interaction and to discover potential markers of infection. Systematic mol. profiling across tissue sections represents a new imaging modality, enabling regiospecific mol. measurements to be made in situ, in both two-dimensional and three-dimensional analyses. Herein, we briefly summarize work that employs MALDI MS to study the pathogenesis of microbial infection.
    18. 18
      Aerts, J. M. F. G.; Artola, M.; van Eijk, M.; Ferraz, M. J.; Boot, R. G. Glycosphingolipids and Infection. Potential New Therapeutic Avenues. Frontiers in Cell and Developmental BiologyFront. Cell Dev. Biol. 2019, 7, 324,  DOI: 10.3389/fcell.2019.00324
      There is no corresponding record for this reference.
    19. 19
      Lopez, P. H.; Schnaar, R. L. Gangliosides in Cell Recognition and Membrane Protein Regulation. Curr. Opin. Struct. Biol. 2009, 19, 549– 557,  DOI: 10.1016/j.sbi.2009.06.001
      19
      Gangliosides in cell recognition and membrane protein regulation
      Lopez, Pablo H. H.; Schnaar, Ronald L.
      Current Opinion in Structural Biology (2009), 19 (5), 549-557CODEN: COSBEF; ISSN:0959-440X. (Elsevier B.V.)
      A review. Gangliosides, sialic acid-bearing glycosphingolipids, are expressed on all vertebrate cells, and are the major glycans on nerve cells. They are anchored to the plasma membrane through their ceramide lipids with their varied glycans extending into the extracellular space. Through sugar-specific interactions with glycan-binding proteins on apposing cells, gangliosides function as receptors in cell-cell recognition, regulating natural killer cell cytotoxicity via Siglec-7, myelin-axon interactions via Siglec-4 (myelin-assocd. glycoprotein), and inflammation via E-selectin. Gangliosides also interact laterally in their own membranes, regulating the responsiveness of signaling proteins including the insulin, epidermal growth factor, and vascular endothelial growth factor receptors. In these ways, gangliosides act as regulatory elements in the immune system, in the nervous system, in metabolic regulation, and in cancer progression.
    20. 20
      Nakayama, H.; Nagafuku, M.; Suzuki, A.; Iwabuchi, K.; Inokuchi, J.-I. The Regulatory Roles of Glycosphingolipid-Enriched Lipid Rafts in Immune Systems. FEBS Lett. 2018, 592, 3921– 3942,  DOI: 10.1002/1873-3468.13275
      There is no corresponding record for this reference.
    21. 21
      Zhang, T.; Waard, A. A. D.; Wuhrer, M.; Spaapen, R. M. The Role of Glycosphingolipids in Immune Cell Functions. Frontiers in Immunology 2019, 10, 90,  DOI: 10.3389/fimmu.2019.00090
      There is no corresponding record for this reference.
    22. 22
      Potapenko, M.; Shurin, G. V.; de León, J. Gangliosides As Immunomodulators. Advances in Experimental Medicine and Biology 2007, 601, 195– 203,  DOI: 10.1007/978-0-387-72005-0_20
      There is no corresponding record for this reference.
    23. 23
      Muggli, T.; Bühr, C.; Schürch, S. Challenges in the Analysis of Gangliosides by LC-MS. CHIMIA 2022, 76, 109,  DOI: 10.2533/chimia.2022.109
      There is no corresponding record for this reference.
    24. 24
      Inokuchi, J. I.; Nagafuku, M.; Ohno, I.; Suzuki, A. Heterogeneity of Gangliosides among T Cell Subsets. Cell. Mol. Life Sci. 2013, 70, 3067– 3075,  DOI: 10.1007/s00018-012-1208-x
      There is no corresponding record for this reference.
    25. 25
      Takahashi, T.; Suzuki, T. Role of Sulfatide in Normal and Pathological Cells and Tissues. J. Lipid Res. 2012, 53, 1437– 1450,  DOI: 10.1194/jlr.R026682
      25
      Role of sulfatide in normal and pathological cells and tissues
      Takahashi, Tadanobu; Suzuki, Takashi
      Journal of Lipid Research (2012), 53 (8), 1437-1450CODEN: JLPRAW; ISSN:0022-2275. (American Society for Biochemistry and Molecular Biology, Inc.)
      A review. Sulfatide is 3-O-sulfogalactosylceramide that is synthesized by two transferases (ceramide galactosyltransferase and cerebroside sulfotransferase) from ceramide and is specifically degraded by a sulfatase (arylsulfatase A). Sulfatide is a multifunctional mol. for various biol. fields including the nervous system, insulin secretion, immune system, hemostasis/thrombosis, bacterial infection, and virus infection. Therefore, abnormal metab. or expression change of sulfatide could cause various diseases. Here, we discuss the important biol. roles of sulfatide in the nervous system, insulin secretion, immune system, hemostasis/thrombosis, cancer, and microbial infections including human immunodeficiency virus and influenza A virus. Our review will be helpful to achieve a comprehensive understanding of sulfatide, which serves as a fundamental target of prevention of and therapy for nervous disorders, diabetes mellitus, immunol. diseases, cancer, and infectious diseases.
    26. 26
      Yu, R. K.; Tsai, Y. T.; Ariga, T.; Yanagisawa, M. Structures, Biosynthesis, and Functions of Gangliosides-an Overview. J. Oleo Sci. 2011, 60, 537– 544,  DOI: 10.5650/jos.60.537
      26
      Structures, biosynthesis, and functions of gangliosides-an overview
      Yu, Robert K.; Tsai, Yi-Tzang; Ariga, Toshio; Yanagisawa, Makoto
      Journal of Oleo Science (2011), 60 (10), 537-544CODEN: JOSOAP; ISSN:1345-8957. (Japan Oil ChemistsÏ Society)
      A review. Gangliosides are sialic acid-contg. glycosphingolipids that are most abundant in the nervous system. Heterogeneity and diversity of the structures in their carbohydrate chains are characteristic hallmarks of these lipids; so far, 188 gangliosides with different carbohydrate structures have been identified in vertebrates. The mol. structural complexity increases manifold if one considers heterogeneity in the lipophilic components. The expression levels and patterns of brain gangliosides are known to change drastically during development. In cells, gangliosides are primarily, but not exclusively, localized in the outer leaflets of plasma membranes and are integral components of cell surface microdomains with sphingomyelin and cholesterol from which they participate in cell-cell recognition, adhesion, and signal transduction. In this brief review, we discuss the structures, metab. and functions of gangliosides.
    27. 27
      AWASTHI, Y. C.; SRIVASTAVA, S. K. STRUCTURE, FUNCTION AND METABOLISM OF GLYCOSPHINGOLIPIDS. Biochemistry of Brain 1980, 1– 20,  DOI: 10.1016/B978-0-08-021345-3.50004-6
      There is no corresponding record for this reference.
    28. 28
      Lunghi, G.; Fazzari, M.; Biase, E. D.; Mauri, L.; Chiricozzi, E.; Sonnino, S. The Structure of Gangliosides Hides a Code for Determining Neuronal Functions. FEBS Open Biol. 2021, 11, 3193,  DOI: 10.1002/2211-5463.13197
      There is no corresponding record for this reference.
    29. 29
      Novaconi, C. R.; Onulov, R.; Serb, A. F.; Sisu, E.; Dinca, N.; Pascariu, M.-C.; Georgescu, M. Assessing Glycosphingolipid Profiles in Human Health and Disease Using Non-Imaging MALDI Mass Spectrometry. Applied Sciences 2023, Vol. 13, Page 9922 2023, 13, 9922,  DOI: 10.3390/app13179922
      There is no corresponding record for this reference.
    30. 30
      Varki, A. Diversity in the Sialic Acids. Glycobiology 1992, 2 (1), 25– 40,  DOI: 10.1093/glycob/2.1.25
      30
      Diversity in the sialic acids
      Varki, Ajit
      Glycobiology (1992), 2 (1), 25-40CODEN: GLYCE3; ISSN:0959-6658.
      A review, with many refs., of the occurrence, structure, biochem., and biol. significance of the diversity of sialic acids in oligosaccharides and sialoglycoproteins. Sialic acid nomenclature is also discussed.
    31. 31
      Ghosh, S. Sialic Acid and Biology of Life: An Introduction. Sialic Acids and Sialoglycoconjugates in the Biology of Life, Health and Disease 2020, 1,  DOI: 10.1016/B978-0-12-816126-5.00001-9
      There is no corresponding record for this reference.
    32. 32
      Ito, E.; Tominaga, A.; Waki, H.; Miseki, K.; Tomioka, A.; Nakajima, K.; Kakehi, K.; Suzuki, M.; Taniguchi, N.; Suzuki, A. Structural Characterization of Monosialo-, Disialo- and Trisialo-Gangliosides by Negative Ion AP-MALDI-QIT-TOF Mass Spectrometry with MSn Switching. Neurochem. Res. 2012, 37, 1315– 1324,  DOI: 10.1007/s11064-012-0735-z
      There is no corresponding record for this reference.
    33. 33
      Barrientos, R. C.; Zhang, Q. Recent Advances in the Mass Spectrometric Analysis of Glycosphingolipidome - A Review. Anal. Chim. Acta 2020, 1132, 134– 155,  DOI: 10.1016/j.aca.2020.05.051
      33
      Recent advances in the mass spectrometric analysis of glycosphingolipidome - A review
      Barrientos, Rodell C.; Zhang, Qibin
      Analytica Chimica Acta (2020), 1132 (), 134-155CODEN: ACACAM; ISSN:0003-2670. (Elsevier B.V.)
      A review. Aberrant expression of glycosphingolipids has been implicated in a myriad of diseases, but the authors' understanding of the strucural diversity, spatial distribution, and biol. function of this class of biomols. remains limited. These challenges partly stem from a lack of sensitive tools that can detect, identify, and quantify glycosphingolipids at the mol. level. Mass spectrometry has emerged as a powerful tool poised to address most of these challenges. Here, the authors review the recent developments in anal. glycosphingolipidomics with an emphasis on sample prepn., mass spectrometry and tandem mass spectrometry-based structural characterization, label-free and labeling-based quantification. The authors also discuss the nomenclature of glycosphingolipids, and emerging technologies like ion mobility spectrometry in differentiation of glycosphingolipid isomers. The intrinsic advantages and shortcomings of each method are carefully critiqued in line with an individual's research goals. Finally, future perspectives on anal. sphingolipidomics are stated, including a need for novel and more sensive methods in isomer sepn., low abundance species detection, and profiling the spatial distribution of glycosphingolipid mol. species in cells and tissues using imaging mass spectrometry.
    34. 34
      Li, H.; Liu, Y.; Wang, Z.; Xie, Y.; Yang, L.; Zhao, Y.; Tian, R. Mass Spectrometry-Based Ganglioside Profiling Provides Potential Insights into Alzheimer’s Disease Development. Journal of Chromatography A 2022, 1676, 463196,  DOI: 10.1016/j.chroma.2022.463196
      There is no corresponding record for this reference.
    35. 35
      Suteanu-Simulescu, A.; Sarbu, M.; Ica, R.; Petrica, L.; Zamfir, A. D. Ganglioside Analysis in Body Fluids by Liquid-Phase Separation Techniques Hyphenated to Mass Spectrometry. Electrophoresis 2023, 44, 501– 520,  DOI: 10.1002/elps.202200229
      There is no corresponding record for this reference.
    36. 36
      Yang, E.; Dufresne, M.; Chaurand, P. Enhancing Ganglioside Species Detection for MALDI-TOF Imaging Mass Spectrometry in Negative Reflectron Mode. Int. J. Mass Spectrom. 2019, 437, 3– 9,  DOI: 10.1016/j.ijms.2017.09.011
      36
      Enhancing ganglioside species detection for MALDI-TOF imaging mass spectrometry in negative reflectron mode
      Yang, Ethan; Dufresne, Martin; Chaurand, Pierre
      International Journal of Mass Spectrometry (2019), 437 (), 3-9CODEN: IMSPF8; ISSN:1387-3806. (Elsevier B.V.)
      Enhanced ganglioside species detection was achieved for matrix assisted laser desorption ionization time-of-flight imaging mass spectrometry (MALDI-TOF IMS) in neg. reflectron mode using a novel sample prepn. protocol that involves washing the tissue in ammonium salt solns. followed by spray depositing ammonium salts and waiting 24 h after sublimation of 1,5-diaminonaphthalene (DAN) before data acquisition. Application of this novel method to normal adult mouse brains led to more than 10-fold increase in total ion intensity in the ganglioside mass range and an increase in the no. of detected sialylated species from 3 to 15, with no apparent delocalization obsd. at 20μm spatial resoln., making it a powerful technique with the potential to provide greater information about gangliosides in numerous biol. contexts.
    37. 37
      Colsch, B.; Jackson, S. N.; Dutta, S.; Woods, A. S. Molecular Microscopy of Brain Gangliosides: Illustrating Their Distribution in Hippocampal Cell Layers. ACS Chem. Neurosci. 2011, 2, 213– 222,  DOI: 10.1021/cn100096h
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      Molecular Microscopy of Brain Gangliosides: Illustrating their Distribution in Hippocampal Cell Layers
      Colsch, Benoit; Jackson, Shelley N.; Dutta, Sucharita; Woods, Amina S.
      ACS Chemical Neuroscience (2011), 2 (4), 213-222CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)
      Gangliosides are amphiphilic mols. found in the outer layer of plasma membranes of all vertebrate cells. They play a major role in cell recognition and signaling and are involved in diseases affecting the central nervous system (CNS). We are reporting the differential distribution of ganglioside species in the rat brain's cerebrum, based on their ceramide assocd. core, and for the first time the presence of acetylation detected by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, which was used to map and image gangliosides with detailed structural information and histol. accuracy. In the hippocampus, localization of the major species GM1, GD1, O-acetylGD1, GT1, and O-acetylGT1 depends on the sphingoid base (d18:1 sphingosine or d20:1 eicosasphingosine) in the mol. layer of the dentate gyrus (ML), which is made up of three distinct layers, the inner mol. layer (IML), which contains sphingosine exclusively, and the middle mol. layer (MML) and the outer mol. layer (OML) where eicosasphingosine is the only sphingoid base. These results demonstrate that there is a different distribution of gangliosides in neuronal axons and dendrites depending on the ceramide core of each layer. GM3, GM2, GD3, and GD2 contain sphingosine predominantly and are mainly present in body cell layers, which are made up of the pyramidal cell layer (Py) and the granular layer of the dentate gyrus (GL), in contrast with GQ1 and the O-acetylated forms of GD1, GT1, and GQ1 gangliosides, which contain both sphingoid bases. However their distribution is based on the sialylated and acetylated oligosaccharide chains in the neuronal cell bodies.
    38. 38
      Harris, A.; Roseborough, A.; Mor, R.; Yeung, K. K.-C.; Whitehead, S. N. Ganglioside Detection from Formalin-Fixed Human Brain Tissue Utilizing MALDI Imaging Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2020, 31, 479– 487,  DOI: 10.1021/jasms.9b00110
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      Ganglioside Detection from Formalin-Fixed Human Brain Tissue Utilizing MALDI Imaging Mass Spectrometry
      Harris, Aaron; Roseborough, A.; Mor, Rahul; Yeung, Ken K.-C.; Whitehead, Shawn N.
      Journal of the American Society for Mass Spectrometry (2020), 31 (3), 479-487CODEN: JAMSEF; ISSN:1879-1123. (American Chemical Society)
      Matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is used to perform mass spectrometric anal. directly on biol. samples providing visual and anatomical spatial information on mols. within tissues. A current obscuration of MALDI-IMS is that it is largely performed on fresh frozen tissue, whereas clin. tissue samples stored long-term are fixed in formalin, and the fixation process is thought to cause signal suppression for lipid mols. Studies have shown that fresh frozen tissue sections applied with an ammonium formate (AF) wash prior to matrix application in the MALDI-IMS procedure display an increase in obsd. signal intensity and sensitivity for lipid mols. detected within the brain while maintaining the spatial distribution of mols. throughout the tissue. In this work, we investigate the viability of formalin-fixed tissue imaging in a clin. setting by comparing MALDI data of fresh frozen and postfixed rat brain samples, along with postfixed human brain samples washed with AF to assess the capabilities of ganglioside anal. in MALDI imaging of formalin-fixed tissue. Results herein demonstrate that MALDI-IMS spectra for gangliosides, including GM1, were significantly enhanced in fresh frozen rat brain, formalin-fixed rat brain, and formalin-fixed human brain samples through the use of an AF wash. Improvements in MALDI-IMS image quality were demonstrated, and the spatial distribution of mols. was retained. Results indicate that this method will allow for the anal. of gangliosides from formalin-fixed clin. samples, which can open addnl. avenues for neurodegenerative disease research.
    39. 39
      Zhang, Y.; Wang, J.; Liu, J.; Han, J.; Xiong, S.; Yong, W.; Zhao, Z. Combination of ESI and MALDI Mass Spectrometry for Qualitative, Semi-Quantitative and in Situ Analysis of Gangliosides in Brain. Sci. Rep. 2016, 6, 1– 11,  DOI: 10.1038/srep25289
      39
      Multivariate time series analysis on the dynamic relationship between Class B notifiable diseases and gross domestic product (GDP) in China
      Zhang, Tao; Yin, Fei; Zhou, Ting; Zhang, Xing-Yu; Li, Xiao-Song
      Scientific Reports (2016), 6 (1), 1-10CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
      The surveillance of infectious diseases is of great importance for disease control and prevention, and more attention should be paid to the Class B notifiable diseases in China. Meanwhile, according to the International Monetary Fund (IMF), the annual growth of Chinese gross domestic product (GDP) would decelerate below 7% after many years of soaring. Under such circumstances, this study aimed to answer what will happen to the incidence rates of infectious diseases in China if Chinese GDP growth remained below 7% in the next five years. Firstly, time plots and cross-correlation matrixes were presented to illustrate the characteristics of data. Then, the multivariate time series (MTS) models were proposed to explore the dynamic relationship between incidence rates and GDP. Three kinds of MTS models, i.e., vector auto-regressive (VAR) model for original series, VAR model for differenced series and error-correction model (ECM), were considered in this study. The rank of error-correction term was taken as an indicator for model selection. Finally, our results suggested that four kinds of infectious diseases (epidemic hemorrhagic fever, pertussis, scarlet fever and syphilis) might need attention in China because their incidence rates have increased since the year 2010.
    40. 40
      Škrášková, K.; Claude, E.; Jones, E. A.; Towers, M.; Ellis, S. R.; Heeren, R. M. A. Enhanced Capabilities for Imaging Gangliosides in Murine Brain with Matrix-Assisted Laser Desorption/Ionization and Desorption Electrospray Ionization Mass Spectrometry Coupled to Ion Mobility Separation. Methods 2016, 104, 69– 78,  DOI: 10.1016/j.ymeth.2016.02.014
      40
      Enhanced capabilities for imaging gangliosides in murine brain with matrix-assisted laser desorption/ionization and desorption electrospray ionization mass spectrometry coupled to ion mobility separation
      Skraskova, Karolina; Claude, Emmanuelle; Jones, Emrys A.; Towers, Mark; Ellis, Shane R.; Heeren, Ron M. A.
      Methods (Amsterdam, Netherlands) (2016), 104 (), 69-78CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)
      The increased interest in lipidomics calls for improved yet simplified methods of lipid anal. Over the past two decades, mass spectrometry imaging (MSI) has been established as a powerful technique for the anal. of mol. distribution of a variety of compds. across tissue surfaces. Matrix-assisted laser desorption/ionization (MALDI) MSI is widely used to study the spatial distribution of common lipids. However, a thorough sample prepn. and necessity of vacuum for efficient ionization might hamper its use for high-throughput lipid anal. Desorption electrospray ionization (DESI) is a relatively young MS technique. In DESI, ionization of mols. occurs under ambient conditions, which alleviates sample prepn. Moreover, DESI does not require the application of an external matrix, making the detection of low mass species more feasible due to the lack of chem. matrix background. However, irresp. of the ionization method, the final information obtained during an MSI expt. is very complex and its anal. becomes challenging. It was shown that coupling MSI to ion mobility sepn. (IMS) simplifies imaging data interpretation. Here we employed DESI and MALDI MSI for a lipidomic anal. of the murine brain using the same IMS-enabled instrument. We report for the first time on the DESI IMS-MSI of multiply sialylated ganglioside species, as well as their acetylated versions, which we detected directly from the murine brain tissue. We show that poly-sialylated gangliosides can be imaged as multiply charged ions using DESI, while they are clearly sepd. from the rest of the lipid classes based on their charge state using ion mobility. This represents a major improvement in MSI of intact fragile lipid species. We addnl. show that complementary lipid information is reached under particular conditions when DESI is compared to MALDI MSI.
    41. 41
      Jackson, S. N.; Colsch, B.; Egan, T.; Lewis, E. K.; Schultz, J. A.; Woods, A. S. Gangliosides’ Analysis by MALDI-Ion Mobility MS. Analyst 2011, 136, 463– 466,  DOI: 10.1039/C0AN00732C
      41
      Gangliosides' analysis by MALDI-ion mobility MS
      Jackson, Shelley N.; Colsch, Benoit; Egan, Thomas; Lewis, Ernest K.; Schultz, J. Albert; Woods, Amina S.
      Analyst (Cambridge, United Kingdom) (2011), 136 (3), 463-466CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
      The combination of ion mobility with matrix-assisted laser desorption/ionization allows for the rapid sepn. and anal. of biomols. in complex mixts. (such as tissue sections and cellular exts.), as isobaric lipid, peptide, and oligonucleotide mol. ions are pre-sepd. in the mobility cell before mass anal. MALDI-IM MS was used to analyze gangliosides, a class of complex glycosphingolipids that has different degrees of sialylation. Both GD1a and GD1b, structural isomers, were studied to see the effects on gas-phase structure depending upon the localization of the sialic acids. A total ganglioside ext. from mouse brain was also analyzed to measure the effectiveness of ion mobility to sep. out the different ganglioside species in a complex mixt.
    42. 42
      Djambazova, K. V.; Dufresne, M.; Migas, L. G.; Kruse, A. R. S.; Plas, R. V. de; Caprioli, R. M.; Spraggins, J. M. MALDI TIMS IMS of Disialoganglioside Isomers─GD1a and GD1b in Murine Brain Tissue. Anal. Chem. 2023, 95, 1176,  DOI: 10.1021/acs.analchem.2c03939
      42
      MALDI TIMS IMS of Disialoganglioside Isomers-GD1a and GD1b in Murine Brain Tissue
      Djambazova, Katerina V.; Dufresne, Martin; Migas, Lukasz G.; Kruse, Angela R. S.; Van de Plas, Raf; Caprioli, Richard M.; Spraggins, Jeffrey M.
      Analytical Chemistry (Washington, DC, United States) (2023), 95 (2), 1176-1183CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Gangliosides are acidic glycosphingolipids, contg. ceramide moieties and oligosaccharide chains with one or more sialic acid residue(s) and are highly diverse isomeric structures with distinct biol. roles. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) enables the untargeted spatial anal. of gangliosides, among other biomols., directly from tissue sections. Integrating trapped ion mobility spectrometry with MALDI IMS allows for the anal. of isomeric lipid structures in situ. Here, we demonstrate the gas-phase sepn. and identification of disialoganglioside isomers GD1a and GD1b that differ in the position of a sialic acid residue, in multiple samples, including a std. mixt. of both isomers, a biol. ext., and directly from thin tissue sections. The unique spatial distributions of GD1a/b (d36:1) and GD1a/b (d38:1) isomers were detd. in rat hippocampus and spinal cord tissue sections, demonstrating the ability to structurally characterize and spatially map gangliosides based on both the carbohydrate chain and ceramide moieties.
    43. 43
      Fernandez-Lima, F. A.; Kaplan, D. A.; Park, M. A. Note: Integration of Trapped Ion Mobility Spectrometry with Mass Spectrometry. Rev. Sci. Instrum. 2011, 82, 126106,  DOI: 10.1063/1.3665933
      43
      Note: Integration of trapped ion mobility spectrometry with mass spectrometry
      Fernandez-Lima, F. A.; Kaplan, D. A.; Park, M. A.
      Review of Scientific Instruments (2011), 82 (12), 126106/1-126106/3CODEN: RSINAK; ISSN:0034-6748. (American Institute of Physics)
      The integration of a trapped ion mobility spectrometer (TIMS) with a mass spectrometer (MS) for complementary fast, gas-phase mobility sepn. prior to mass anal. (TIMS-MS) is described. The ion transmission and mobility sepn. are discussed as a function of the ion source condition, bath gas velocity, anal. scan speed, RF ion confinement, and downstream ion optical conditions. TIMS mobility resoln. depends on the anal. scan speed and the bath gas velocity, with the unique advantage that the IMS sepn. can be easily tuned from high speed (≈25 ms) for rapid anal. to slower scans for higher mobility resoln. (R >80). (c) 2011 American Institute of Physics.
    44. 44
      Fernandez-Lima, F.; Kaplan, D. A.; Suetering, J.; Park, M. A. Gas-Phase Separation Using a Trapped Ion Mobility Spectrometer. International journal for ion mobility spectrometry 2011, 14, 93,  DOI: 10.1007/s12127-011-0067-8
      There is no corresponding record for this reference.
    45. 45
      Spraggins, J. M.; Djambazova, K. V.; Rivera, E. S.; Migas, L. G.; Neumann, E. K.; Fuetterer, A.; Suetering, J.; Goedecke, N.; Ly, A.; Plas, R. V. D.; Caprioli, R. M. High-Performance Molecular Imaging with MALDI Trapped Ion-Mobility Time-of-Flight (TimsTOF) Mass Spectrometry. Anal. Chem. 2019, 91, 14552– 14560,  DOI: 10.1021/acs.analchem.9b03612
      45
      High-Performance Molecular Imaging with MALDI Trapped Ion-Mobility Time-of-Flight (timsTOF) Mass Spectrometry
      Spraggins, Jeffrey M.; Djambazova, Katerina V.; Rivera, Emilio S.; Migas, Lukasz G.; Neumann, Elizabeth K.; Fuetterer, Arne; Suetering, Juergen; Goedecke, Niels; Ly, Alice; Van de Plas, Raf; Caprioli, Richard M.
      Analytical Chemistry (Washington, DC, United States) (2019), 91 (22), 14552-14560CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Imaging mass spectrometry (IMS) enables the spatially targeted mol. assessment of biol. tissues at cellular resolns. New developments and technologies are essential for uncovering the mol. drivers of native physiol. function and disease. Instrumentation must maximize spatial resoln., throughput, sensitivity, and specificity, because tissue imaging expts. consist of thousands to millions of pixels. Here, the authors report the development and application of a matrix-assisted laser desorption/ionization (MALDI) trapped ion mobility spectrometry imaging platform. This prototype MALDI timsTOF instrument is capable of 10 μm spatial resolns. and 20 pixels/s throughput mol. imaging. The MALDI source utilizes a Bruker SmartBeam 3-D laser system that can generate a square burn pattern of <10 × 10 μm at the sample surface. General image performance was assessed using murine kidney and brain tissues and demonstrate that high spatial resoln. imaging data can be generated rapidly with mass measurement errors < 5 ppm and ∼40,000 resolving power. Initial TIMS-based imaging expts. were performed on whole body mouse pup tissue demonstrating the sepn. of closely isobaric [PC(32:0)+Na]+ and [PC(34:3)+H]+ (3 mDa mass difference) in the gas-phase. The authors have shown that the MALDI timsTOF platform can maintain reasonable data acquisition rates (>2 pixels/s) while providing the specificity necessary to differentiate components in complex mixts. of lipid adducts. The combination of high spatial resoln. and throughput imaging capabilities with high-performance TIMS sepns. provides a uniquely tunable platform to address many challenges assocd. with advanced mol. imaging applications.
    46. 46
      Michelmann, K.; Silveira, J. A.; Ridgeway, M. E.; Park, M. A. Fundamentals of Trapped Ion Mobility Spectrometry. J. Am. Soc. Mass Spectrom. 2015, 26, 14– 24,  DOI: 10.1007/s13361-014-0999-4
      46
      Fundamentals of Trapped Ion Mobility Spectrometry
      Michelmann, Karsten; Silveira, Joshua A.; Ridgeway, Mark E.; Park, Melvin A.
      Journal of the American Society for Mass Spectrometry (2015), 26 (1), 14-24CODEN: JAMSEF; ISSN:1044-0305. (Springer)
      Trapped ion mobility spectrometry (TIMS) is a relatively new gas-phase sepn. method that was coupled to quadrupole orthogonal acceleration time-of-flight mass spectrometry. The TIMS analyzer is a segmented radiofrequency ion guide wherein ions are mobility-analyzed using an elec. field that holds ions stationary against a moving gas, unlike conventional drift tube ion mobility spectrometry where the gas is stationary. Ions are initially trapped, and subsequently eluted from the TIMS analyzer over time according to their mobility (K). Though TIMS has achieved a high level of performance (R > 250) in a small device (<5 cm) using modest operating potentials (<300 V), a proper theory has yet to be produced. Here, the authors develop a quant. theory for TIMS via math. derivation and simulations. A 1-dimensional anal. model, used to predict the transit time and theor. resolving power, is described. Theor. trends are in agreement with exptl. measurements performed as a function of K, pressure, and the axial elec. field scan rate. The linear dependence of the transit time with 1/K provides a fundamental basis for detn. of reduced mobility or collision cross section values by calibration. The quant. description of TIMS provides an operational understanding of the analyzer, outlines the current performance capabilities, and provides insight into future avenues for improvement. [Figure not available: see fulltext.].
    47. 47
      Silveira, J. A.; Michelmann, K.; Ridgeway, M. E.; Park, M. A. Fundamentals of Trapped Ion Mobility Spectrometry Part II: Fluid Dynamics. J. Am. Soc. Mass Spectrom. 2016, 27, 585– 595,  DOI: 10.1007/s13361-015-1310-z
      47
      Fundamentals of Trapped Ion Mobility Spectrometry Part II: Fluid Dynamics
      Silveira, Joshua A.; Michelmann, Karsten; Ridgeway, Mark E.; Park, Melvin A.
      Journal of the American Society for Mass Spectrometry (2016), 27 (4), 585-595CODEN: JAMSEF; ISSN:1044-0305. (Springer)
      Trapped ion mobility spectrometry (TIMS) is a high resoln. (R up to ∼300) sepn. technique which uses an elec. field to hold ions stationary against a moving gas. Recently, an anal. model for TIMS was derived and, in part, exptl. verified. A central, but not yet fully explored, model component involves fluid dynamics at work. This work characterized fluid dynamics in TIMS using simulations and ion mobility expts. Results indicated subsonic laminar flow develops in the analyzer, with pressure-dependent gas velocities from ∼120 to 170 m/s measured at the ion elution position. A key philosophical question is: how can mobility be measured in a dynamic system where the gas is expanding and its velocity is changing. The authors previously noted that anal. useful work is primarily done on ions as they traverse the elec. field gradient plateau in the analyzer. This work showed the position-dependent change in gas velocity on the plateau is balanced by a change in pressure and temp., ultimately resulting in a near, position-independent drag force. Since the drag force and related variables are nearly const., they allow for the use of relatively simple equations to describe TIMS behavior. Nonetheless, a more comprehensive model, which accounts for the spatial dependence of flow variables, was derived. Exptl. resolving power trends closely agreed with the theor. dependence of the drag force, thereby validating another principal component of TIMS theory.
    48. 48
      Ridgeway, M. E.; Lubeck, M.; Jordens, J.; Mann, M.; Park, M. A. Trapped Ion Mobility Spectrometry: A Short Review. Int. J. Mass Spectrom. 2018, 425, 22– 35,  DOI: 10.1016/j.ijms.2018.01.006
      48
      Trapped ion mobility spectrometry: A short review
      Ridgeway, Mark E.; Lubeck, Markus; Jordens, Jan; Mann, Mattias; Park, Melvin A.
      International Journal of Mass Spectrometry (2018), 425 (), 22-35CODEN: IMSPF8; ISSN:1387-3806. (Elsevier B.V.)
      Trapped ion mobility spectrometry (TIMS) hybridized with mass spectrometry (MS) is a relatively recent advance in the field of ion mobility mass spectrometry (IMMS). The basic idea behind TIMS is the reversal of the classic drift cell analyzer. Rather than driving ions through a stationary gas, as in a drift cell, TIMS holds the ions stationary in a moving column of gas. This has the immediate advantage that the phys. dimension of the analyzer can be small (∼5 cm) whereas the anal. column of gas - the column that flows past during the course of an anal. - can be large (as much as 10 m) and user defined. In the years since the first publication, TIMS has proven to be a highly versatile alternative to drift tube ion mobility achieving high resolving power (R ∼ 300), duty cycle (100%), and efficiency (∼80%). In addn. to its basic performance specifications, the flexibility of TIMS allows it to be adapted to a variety of applications. This is highlighted particularly by the PASEF (parallel accumulation serial fragmentation) workflow, which adapts TIMS-MS to the shotgun proteomics application. In this brief review, the general operating principles, theory, and a no. of TIMS-MS applications are summarized.
    49. 49
      Djambazova, K.; Klein, D. R.; Migas, L. G.; Neumann, E. K.; Rivera, E. S.; Plas, R. V. de; Caprioli, R. M.; Spraggins, J. M. Resolving the Complexity of Spatial Lipidomics Using MALDI TIMS Imaging Mass Spectrometry. Anal. Chem. 2020, 92 (19), 13290– 13297,  DOI: 10.1021/acs.analchem.0c02520
      49
      Resolving the Complexity of Spatial Lipidomics Using MALDI TIMS Imaging Mass Spectrometry
      Djambazova, Katerina V.; Klein, Dustin R.; Migas, Lukasz G.; Neumann, Elizabeth K.; Rivera, Emilio S.; Van de Plas, Raf; Caprioli, Richard M.; Spraggins, Jeffrey M.
      Analytical Chemistry (Washington, DC, United States) (2020), 92 (19), 13290-13297CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Lipids are a structurally diverse class of mols. with important biol. functions including cellular signaling and energy storage. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows for direct mapping of biomols. in tissues. Fully characterizing the structural diversity of lipids remains a challenge due to the presence of isobaric and isomeric species, which greatly complicates data interpretation when only m/z information is available. Integrating ion mobility sepns. aids in deconvoluting these complex mixts. and addressing the challenges of lipid IMS. Here, the authors demonstrate that a MALDI quadrupole time-of-flight (Q-TOF) mass spectrometer with trapped ion mobility spectrometry (TIMS) enables a >250% increase in the peak capacity during IMS expts. MALDI TIMS-MS sepn. of lipid isomer stds., including sn backbone isomers, acyl chain isomers, and double-bond position and stereoisomers, is demonstrated. As a proof of concept, in situ sepn. and imaging of lipid isomers with distinct spatial distributions were performed using tissue sections from a whole-body mouse pup.
    50. 50
      Hussein, M. R. Mucocutaneous Splendore-Hoeppli Phenomenon. Journal of Cutaneous Pathology 2008, 35, 979– 988,  DOI: 10.1111/j.1600-0560.2008.01045.x
      There is no corresponding record for this reference.
    51. 51
      Martínez-Girón, R.; Pantanowitz, L. Splendore-Hoeppli” Phenomenon. Diagnostic Cytopathology 2020, 48, 1316– 1317,  DOI: 10.1002/dc.24512
      There is no corresponding record for this reference.
    52. 52
      de Carvalho, T. P.; Eckstein, C.; de Moura, L. L.; Heleno, N. V. R.; da Silva, L. A.; dos Santos, D. O.; de Souza, L. d. R.; Oliveira, A. R.; Xavier, R. G. C.; Thompson, M.; Silva, R. O. S.; Santos, R. L. Staphylococcus Aureus-Induced Pyogranulomatous Dermatitis, Osteomyelitis, and Meningitis with Splendore-Hoeppli Reaction in a Cat Coinfected with the Feline Leukemia Virus and Leishmania Sp. Braz J Vet Pathol 2022, 15, 31– 37,  DOI: 10.24070/bjvp.1983-0246.v15i1p31-37
      There is no corresponding record for this reference.
    53. 53
      Rivera, E. S.; Weiss, A.; Migas, L. G.; Freiberg, J. A.; Djambazova, K. V.; Neumann, E. K.; Plas, R. V. de; Spraggins, J. M.; Skaar, E. P.; Caprioli, R. M. Imaging Mass Spectrometry Reveals Complex Lipid Distributions across Staphylococcus Aureus Biofilm Layers. J. Mass Spectrom. Adv. Clin. Lab 2022, 26, 36– 46,  DOI: 10.1016/j.jmsacl.2022.09.003
      50
      Imaging mass spectrometry reveals complex lipid distributions across Staphylococcus aureus biofilm layers
      Rivera, Emilio S.; Weiss, Andy; Migas, Lukasz G.; Freiberg, Jeffrey A.; Djambazova, Katerina V.; Neumann, Elizabeth K.; Van de Plas, Raf; Spraggins, Jeffrey M.; Skaar, Eric P.; Caprioli, Richard M.
      Journal of Mass Spectrometry and Advances in the Clinical Lab (2022), 26 (), 36-46CODEN: JMSAC5; ISSN:2667-145X. (Elsevier B.V.)
      Although Staphylococcus aureus is the leading cause of biofilm-related infections, the lipidomic distributions within these biofilms is poorly understood. Here, lipidomic mapping of S. aureus biofilm cross-sections was performed to investigate heterogeneity between horizontal biofilm layers. S. aureus biofilms were grown statically, embedded in a mixt. of CM-cellulose/gelatin, and prepd. for downstream matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Trapped ion mobility spectrometry (TIMS) was also applied prior to mass anal. Implementation of TIMS led to a ∼ threefold increase in the no. of lipid species detected. Washing biofilm samples with ammonium formate (150 mM) increased signal intensity for some bacterial lipids by as much as tenfold, with minimal disruption of the biofilm structure. MALDI TIMS IMS revealed that most lipids localize primarily to a single biofilm layer, and species from the same lipid class such as cardiolipins CL(57:0) - CL(66:0) display starkly different localizations, exhibiting between 1.5 and 6.3-fold intensity differences between layers (n = 3, p < 0.03). No horizontal layers were obsd. within biofilms grown anaerobically, and lipids were distributed homogenously. High spatial resoln. anal. of S. aureus biofilm cross-sections by MALDI TIMS IMS revealed stark lipidomic heterogeneity between horizontal S. aureus biofilm layers demonstrating that each layer was molecularly distinct. Finally, this workflow uncovered an absence of layers in biofilms grown under anaerobic conditions, possibly indicating that oxygen contributes to the obsd. heterogeneity under aerobic conditions. Future applications of this workflow to study spatially localized mol. responses to antimicrobials could provide new therapeutic strategies.
    54. 54
      Good, C. J.; Butrico, C. E.; Colley, M. E.; Gibson-Corley, K. N.; Cassat, J. E.; Spraggins, J. M.; Caprioli, R. M. In Situ Lipidomics of Staphylococcus Aureus Osteomyelitis Using Imaging Mass Spectrometry. bioRxiv , 2023.  DOI: 10.1101/2023.12.01.569690 .
      There is no corresponding record for this reference.
    55. 55
      Yohe, H. C.; Macala, L. J.; Giordano, G.; McMurray, W. J. GM1b and GM1b-GalNAc: Major Gangliosides of Murine-Derived Macrophage-like WEHI-3 Cells. Biochimica et Biophysica Acta (BBA) - Biomembranes 1992, 1109, 210– 217,  DOI: 10.1016/0005-2736(92)90085-Z
      There is no corresponding record for this reference.
    56. 56
      Sarbu, M.; Clemmer, D. E.; Zamfir, A. D. Ion Mobility Mass Spectrometry of Human Melanoma Gangliosides. Biochimie 2020, 177, 226– 237,  DOI: 10.1016/j.biochi.2020.08.011
      101
      Ion mobility mass spectrometry of human melanoma gangliosides
      Sarbu, Mirela; Clemmer, David E.; Zamfir, Alina D.
      Biochimie (2020), 177 (), 226-237CODEN: BICMBE; ISSN:0300-9084. (Elsevier Masson SAS)
      Malignant melanoma is an aggressive type of skin cancer, rarely detected in the early stages. Various sets of methods and techniques, including dermatoscopical inspection of the "ABCDE" signs of the lesion, imaging techniques or microscopical, immunohistochem. and serol. biomarkers are available and used nowadays to diagnose malignant melanoma. To date, different biomarkers were proposed for melanoma, but only a few, including circulating proteins, such as lactate dehydrogenase, mol. and metabolite biomarkers, have reached clin. applications. Gangliosides represent an emerging class, being used as tumor markers and targets of antibody therapy in melanomas, based on their elevated abundance in melanoma, esp. of GM3 and GD3, when compared with the corresponding normal tissues. The conjunction of mass spectrometry (MS) with ion mobility sepn. (IMS) demonstrated an elevated potential in detection and identification of low abundant components, with biomarker role, in extremely complex biol. mixts. Therefore, here, a native ganglioside ext. originating from human melanoma was investigated for the first time by IMS MS to provide the first profiling of gangliosides in this type of cancer. The present approach revealed the high incidence of species belonging to GD3 and GM3 classes, as well as of de-N-acetyl GM3 (d-GM3) and de-N-acetyl GD3 (d-GD3), characteristic for human melanoma. Addnl., the structure of two mols. characterized by shorter glycan chains assocd. to melanoma, were investigated in detail. The present approach brings valuable data related to this type of cancer, completing the existing inventory of melanoma-assocd. biomarkers and opens new directions for further research in this field.
    57. 57
      Metelmann, W.; Vukelić, Ž.; Peter-Katalinić, J. Nano-Electrospray Ionization Time-of-Flight Mass Spectrometry of Gangliosides from Human Brain Tissue. J. Mass Spectrom. 2001, 36, 21– 29,  DOI: 10.1002/jms.100
      102
      Nano-electrospray ionization time-of-flight mass spectrometry of gangliosides from human brain tissue
      Metelmann, Wolfgang; Vukelic, Zeljka; Peter-Katalinic, Jasna
      Journal of Mass Spectrometry (2001), 36 (1), 21-29CODEN: JMSPFJ; ISSN:1076-5174. (John Wiley & Sons Ltd.)
      A general approach for the detection and structural elucidation of brain ganglioside species GM1, GD1 and GT1 by nano-electrospray ionization quadrupole time-of-flight (nanoESI-QTOF) mass spectrometry (MS), using combined data from MS and MS/MS anal. of isolated native ganglioside fractions in neg. ion mode and their permethylated counterparts in the pos. ion mode is presented. This approach was designed to detect and sequence gangliosides present in preparatively isolated ganglioside fractions from pathol. brain samples available in only very limited amts. In these fractions mixts. of homolog and isobaric structures are present, depending on the ceramide compn. and the position of the sialic acid attachment site. The interpretation of data for the entire sequence, derived from A, B, C and Y ions by nanoESI-QTOFMS/MS in the neg. ion mode of native fractions, can be compromised by ions arising from double and triple internal cleavages. To distinguish between isobaric carbohydrate structures in gangliosides, such as monosialogangliosides GM1a and GM1b, disialogangliosides GD1a, GD1b and GD1c or trisialogangliosides GT1b, GT1c and GT1d, the samples were analyzed after permethylation in the pos. ion nanoESI-QTOFMS/MS mode, providing set of data, which allows a clear distinction for assignment of outer and inner fragment ions according to their m/z values. The fragmentation patterns from native gangliosides obtained by low-energy collision induced dissocn. (CID) by nanoESI-QTOF show common behavior and follow inherent rules. The combined set of data from the neg. and pos. ion mode low-energy CID can serve for the detection of structural isomers in mixts., and to trace new, not previously detected, components.
    58. 58
      Schnaar, R. L.; Sandhoff, R.; Tiemeyer, M.; Kinoshita, T. Glycosphingolipids. In Essentials of Glycobiology ; 2022.  DOI: 10.1101/glycobiology.4e.11 .
      There is no corresponding record for this reference.
    59. 59
      Ryan, J. L.; Yohe, H. C.; Cuny, C. L.; Macala, L. J.; Saito, M.; Mcmurray, W. J. The Presence of Sialidase-Sensitive Sialosylgangliotetraosyl Ceramide (GM1b) in Stimulated Murine Macrophages. Deficiency of GM1b in Escherichia Coli-Activated Macrophages from the C3H/HeJ Mouse. J. Immunol. 1991, 146, 1900– 1908,  DOI: 10.4049/jimmunol.146.6.1900
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jasms.4c00089.

    • Ganglioside synthesis pathway; average mass spectrum of control and infected mouse kidney section; ion mobility heat map of 10 DPI mouse kidney average mass spectrum; table summarizing matrix deposition parameters; table summarizing characteristic fragment ions used for ganglioside identification; tables and figures of gangliosides identified in a 10 DPI mouse kidney section, including GM3, GM2, GM1, GalNAc- and extended series GM1b, and GD1; on-tissue fragmentation of GM2 and GalNAc-GM1b; tables listing ganglioside isomers identified in 10 DPI mouse kidney, including GM1a and GM1b, and NeuAc-tCer and NeuGc-dCer; on-tissue fragmentation of a- and o-series ganglioside isomers (m/z 1626.95); molecular structures of GM1 isomers detected at m/z 1532.83; detailed on-tissue fragmentation of m/z 1532.83 (PDF)


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