Spatial Multiomics of Lipids, N-Glycans, and Tryptic Peptides on a Single FFPE Tissue Section

Mass spectrometry imaging (MSI) is an emerging technology that is capable of mapping various biomolecules within their native spatial context, and performing spatial multiomics on formalin-fixed paraffin-embedded (FFPE) tissues may further increase the molecular characterization of pathological states. Here we present a novel workflow which enables the sequential MSI of lipids, N-glycans, and tryptic peptides on a single FFPE tissue section and highlight the enhanced molecular characterization that is offered by combining the multiple spatial omics data sets. In murine brain and clear cell renal cell carcinoma (ccRCC) tissue, the three molecular levels provided complementary information and characterized different histological regions. Moreover, when the spatial omics data was integrated, the different histopathological regions of the ccRCC tissue could be better discriminated with respect to the imaging data set of any single omics class. Taken together, these promising findings demonstrate the capability to more comprehensively map the molecular complexity within pathological tissue.


Supplementary Figure 2.
Spatial multi-omics of lipids, N-glycans, and tryptic peptides on four different ccRCC tissue sections. The spatial distribution and the relative intensity of four example bioanalytes for each molecular class is highlighted. Regarding the lipids, the m/z of the [M-H] ions related to PI(18:0), PE(22:4), PA(18:1/18:0), and PA(16:0/20:4) are reported. Regarding the N-glycans, the m/z of the [M+Na] ions related to Hex8HexNAc6, Hex6HexNAc4, Hex6HexNAc5, and Hex9HexNAc2 are reported. Regarding the tryptic peptides, the m/z of the [M+H] ions of related to MOES (Moesin), TGF1 (Transforming growth factor 1), HBA (Haemoglobin subunit alpha), and ANXA2 (Annexin A2) are reported. In particular, the tissue distribution of these features serves to highlight the capacity of the different molecular classes to underline the distinct histopathological regions.

Supplementary Figure 3.
Haematoxylin and Eosin staining of the ccRCC section used for the statistical analysis. The regions of interest (ROIs) considered in the analysis are highlighted: Pseudocapsule; medulla; leukocytes; TILs (tumour infiltrating leukocytes); G2-G3; G2. Pseudocapsule and medulla were included in the "inflamed tissue" region. G2-G3 and G2 were included in the "Tumour" region. PCA score charts generated from the lipid (A), N-glycan (B), and tryptic peptide (C) datasets, plotting PC1 (x axis) vs PC3 (y axis). A legend with the four major histopathological groups is provided (D). 95% confidence intervals are highlighted by their respective background colour.

Supplementary Figure 6.
The top ten contributing variables (features) to the separation observed along the first three components of the PCA analysis for the lipid (A), N-glycan (B), tryptic peptide (C), and multi-omic datasets (D). The dashed red line indicates a one percent contribution level.

Supplementary Table 1.
List of m/z features included in the curated mass list, identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) following extraction from ccRCC tissue. Accurate mass, ID, and retention time (in minutes) for each feature are reported.

Supplementary Figures
Supplementary Figure 1. Average MALDI mass spectra of lipids, N-glycans, and tryptic peptides in murine brain tissue. The number of peaks per spectrum (p) is indicated. On the bottom, the number of common peaks for each analyte class is reported.

Supplementary Figure 2.
Spatial multi-omics of lipids, N-glycans, and tryptic peptides on four different ccRCC tissue sections. The spatial distribution and the relative intensity of four example bioanalytes for each molecular class is Haematoxylin and Eosin staining of the ccRCC section used for the statistical analysis. The regions of interest (ROIs) considered in the analysis are highlighted: Pseudocapsule; medulla; leukocytes; TILs (tumour infiltrating leukocytes); G2-G3; G2. Pseudocapsule and medulla were included in the "inflamed tissue" region. G2-G3 and G2 were included in the "Tumour" region.

Supplementary Figure 5.
PCA score charts generated from the lipid (A), N-glycan (B), and tryptic peptide (C) datasets, plotting PC1 (x axis) vs PC3 (y axis). A legend with the four major histopathological groups is provided (D). 95% confidence intervals are highlighted by their respective background colour.

Supplementary Figure 6.
The top ten contributing variables (features) to the separation observed along the first three components of the PCA analysis for the lipid (A), N-glycan (B), tryptic peptide (C), and multiomic datasets (D). The dashed red line indicates a one percent contribution level. List of m/z features included in the curated mass list, identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) following extraction from ccRCC tissue. Accurate mass, ID, and retention time (in minutes) for each feature are reported.