Chemical Proteomics Strategies for Analyzing Protein Lipidation Reveal the Bacterial O-Mycoloylome

Protein lipidation dynamically controls protein localization and function within cellular membranes. A unique form of protein O-fatty acylation in Corynebacterium, termed protein O-mycoloylation, involves the attachment of mycolic acids—unusually large and hydrophobic fatty acids—to serine residues of proteins in these organisms’ outer mycomembrane. However, as with other forms of protein lipidation, the scope and functional consequences of protein O-mycoloylation are challenging to investigate due to the inherent difficulties of enriching and analyzing lipidated peptides. To facilitate the analysis of protein lipidation and enable the comprehensive profiling and site mapping of protein O-mycoloylation, we developed a chemical proteomics strategy integrating metabolic labeling, click chemistry, cleavable linkers, and a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method employing LC separation and complementary fragmentation methods tailored to the analysis of lipophilic, MS-labile O-acylated peptides. Using these tools in the model organism Corynebacterium glutamicum, we identified approximately 30 candidate O-mycoloylated proteins, including porins, mycoloyltransferases, secreted hydrolases, and other proteins with cell envelope-related functions—consistent with a role for O-mycoloylation in targeting proteins to the mycomembrane. Site mapping revealed that many of the proteins contained multiple spatially proximal modification sites, which occurred predominantly at serine residues surrounded by conformationally flexible peptide motifs. Overall, this study (i) discloses the putative protein O-mycoloylome for the first time, (ii) yields new insights into the undercharacterized proteome of the mycomembrane, which is a hallmark of important pathogens (e.g., Corynebacterium diphtheriae, Mycobacterium tuberculosis), and (iii) provides generally applicable chemical strategies for the proteomic analysis of protein lipidation.


SI Table of Contents* Supplementary tables _____________________________________________ S3
Table S1.List of strains used in this study _____________________________ S3 Table S2.List of plasmids used in this study ___________________________ S4 Table S3.List of primers used in this study ____________________________ S5 Tables S4-S11.LC-MS/MS raw and curated data _______________________ * Tables S12-S13.Figures 3E and 3F   Supplementary discussion of Figure S1.Treatment of Cg cells with 4% SDS at 100 °C for 5 min was previously reported to extract cell envelope-associated proteins, [5][6][7] which we predicted would serve as a convenient and efficient method for extraction of labeled proteins.To test this method, we labeled Cg WT with O-AlkTMM, then performed three successive extractions under these conditions (4% SDS at 100 °C for 5 min), click-labeled proteins with azido-488, and analyzed the proteins.We found that fluorescently labeled, putatively O-mycoloylated proteins were mainly present in the first two rounds of extraction (lanes 1-4), with minimal total protein or fluorescence signal in the third extraction (lanes 5 and 6).To stringently test whether any labeled proteins remained, the pellets were subjected three times to treatment with lysozyme, bead beating, and extraction with 2% SDS at 60 °C for 2 h, and the protein extracts were processed and visualized as just described.We found that, while significant additional total protein was extracted from the remaining pellets, there was virtually no fluorescence in those proteins (lanes 7-12).Thus, 1-2 extractions of Cg cells using 4% SDS at 100 °C for 5 min is sufficient to efficiently extract all O-AlkTMM-labeled proteins, while also serving as a partial enrichment step for these proteins.

Reagents
The supernatant, expected to contain cell wall-associated proteins, was collected by centrifugation at 13,000 xg for 15 mins at 4 °C.The extraction was repeated twice, and the clear supernatants were combined.This method was used for protein extraction except otherwise stated.
Protein precipitation using chloroform/methanol/water. One part of the alkynelabeled protein extract was added to four parts of ice-cold methanol and one part of ice-cold chloroform and vortexed briefly.Three parts of water were added, vortexed briefly, and centrifuged at 18,000 xg for 2-10 mins at 4°C.The top aqueous layer was carefully removed without disturbing the protein disc at the interface layer.Three parts of ice-cold methanol were added, vortexed briefly, and centrifuged at 18,000 xg for 2-10 mins at 4°C to pellet the proteins.
The supernatant was discarded, and the precipitated proteins were air-dried briefly and resuspended in resuspension buffer (Milli-Q water containing 0.5% SDS and 0.05% LDAO).S3.To construct pCRD206 derivatives, 500bp-1kb fragments encoding homology regions upstream and downstream of the gene of interest were amplified and assembled onto pCRD206 3 through ITA.The pEMH25 empty vector was constructed by first replacing lacI/Ptac/eGFP from the published pTGR5 vector 4 with the Psod promoter native to C. glutamicum (including the native RBS) through ITA.This kanamycin resistance cassette on the resulting was replaced with a chloramphenicol resistance cassette using ITA.Finally, Q5 mutagenesis was used to delete the region downstream of the Psod promoter containing a 6x His tag.porB was amplified from MB001 genomic DNA and cloned onto a pEMH25 derivative containing a 6x His tagged construct via ITA.Site-directed mutagenesis of porB-6xHis was performed using the KLD mutagenesis mix (NEB), following manufacturer specifications.All plasmid constructs were confirmed by Sanger sequencing.

Copper-catalyzed azide-alkyne cycloaddition (CuAAC) conditions.
Strain construction.C. glutamicum competent cells were prepared as previously described. 11,12 ene deletion in C. glutamicum was performed using the temperature-sensitive plasmid pCRD206 with sacB counterselection. 3 were removed using chloroform/methanol/water precipitation.The proteins were air-dried briefly and dissolved in resuspension buffer, then equal amounts of each sample were resolved using SDS-PAGE and analyzed using in-gel fluorescence scanning and Coomassie staining.fluorescence intensity (MFI) values were collected for 50,000 events at an event rate of 500-1,000 events/sec.The experiments were performed in triplicate.

Click chemistry-mediated affinity enrichment using Az-T-B
Protein labeling and extraction.500 mL cultures of Cg type strain 534 at an OD600 of 0.3 were treated with O-AlkTMM to a final concentration of 0.4 mM, or DMSO control, and incubated for 16 h at 30 °C (for LC-MS/MS Study 1, n=4 replicates for both probe-treated and control conditions).For the Az-T-B enrichment study, proteins were extracted through a twostep method of (i) chloroform/methanol extraction and (ii) bead beating in resuspension buffer, then extracted proteins from each step were combined for subsequent analysis as follows.After the O-AlkTMM treatment step, the Cg cells were harvested, washed, air-dried briefly, resuspended in 120 mL of chloroform/methanol (2:1), and stirred for 10 h at room temperature.
The cells were centrifuged at 5,000 xg for 10 min at room temperature, and the supernatants were saved.Next, the cell pellets were resuspended in 120 mL of chloroform/methanol (1:2), stirred at room temperature for 10 h, and pelleted by centrifugation, then the resulting cell pellets and the supernatants were saved.The supernatants from both chloroform/methanol extractions

LC-MS/MS analysis of peptides generated from Az-T-B enrichment (LC-MS/MS Study 1)
Protein digestion and LC-MS/MS analysis.The above method for click chemistrymediated affinity enrichment of O-AlkTMM-labeled proteins using Az-T-B was carried out.Gel bands were digested in-gel, according to Shevchenko et al., 14 with modifications.Briefly, gel bands were washed with 100 mM ammonium bicarbonate and dehydrated using 100% acetonitrile (ACN).Sequencing grade modified trypsin was prepared to 0.01 µg/µL in 50 mM ammonium bicarbonate, and ~100 µL of this was added to each gel band to submerge the gel completely.Bands were then incubated at 37 °C overnight.Peptides were extracted from the gel by water bath sonication in a 60% ACN/1% trifluoroacetic acid (TFA) solution and vacuum dried to ~2 µL.The samples were resuspended to 20 µL using 2% ACN/0.1% TFA.Next, 5 µL was injected automatically using a Thermo EASYnLC 1200 onto a Thermo Acclaim PepMap RSLC 0.1 mm x 20 mm C18 trapping column and washed for ~5 min with buffer A. Bound peptides were then eluted onto a Thermo Acclaim PepMap RSLC 0.075 mm x 500 mm C18 resolving column over 35 min with a gradient of 8% B to 40% B in 24 min, ramping to 90% B in 1 min and held at 90% B for the duration of the run at a constant flow rate of 300 nL/min.Buffer A = 99.9%water/0.1% formic acid, Buffer B = 80% ACN/0.1% formic acid/19.9%H2O.The column's temperature was maintained at a constant temperature of 50 °C using an integrated column oven (PRSO-V2, Sonation GmbH, Biberach, Germany).Eluted peptides were sprayed into a ThermoScientific Q-Exactive HF-X mass spectrometer using a FlexSpray spray ion source.Survey scans were taken in the Orbitrap (60,000 resolution, determined at m/z 200).
The top fifteen ions in each survey scan were subjected to automatic higher energy collisioninduced dissociation (HCD) with fragment spectra acquired at 15,000 resolution.

Data analysis.
The resulting MS/MS spectra were converted to peak lists using MaxQuant, v1.6.3.4,and searched against a protein database containing all Cg sequences appended with common laboratory contaminants (downloaded 2018-06-12 from www.ncbi.nlm.nih.gov and www.thegpm.org,respectively) using the Andromeda search algorithm, a part of the MaxQuant environment.The search output was then analyzed using Scaffold Q+S, v4.10.0, to probabilistically validate protein identifications.Assignments validated using the Scaffold 1% FDR confidence filter are considered true.Mascot parameters for all databases were as follows: allow up to 2 missed tryptic sites; Fixed modification of and control conditions; for sample preparation for LC-MS/MS Study 3, both wild-type and Δcmt1 were used and n=4 for wild-type/probe-treated condition and n=3 for all control conditions).As described in the general procedures, protein extracts were collected, precipitated in chloroform/methanol/water, and resuspended in resuspension buffer.The resuspended proteins were reacted with Az-DADPS-B (100 µM) under CuAAC conditions, and excess unreacted reagents were removed using chloroform/methanol/water precipitation.Biotinylated proteins were then subjected to procedures for protein-level enrichment (Study 2) or peptide-level enrichment (Study 3) as follows.

Protein-level enrichment (LC-MS/MS Study 2). ~1 mg of biotinylated protein extracts
from probe-treated and untreated Cg wild type prepared as described above were added to 100 µL of settled pre-cleaned streptavidin beads and incubated for 2 h at room temperature with endover-end rotation.The beads were centrifuged at 2,000 xg for 1 min, and then the supernatants were discarded.The beads were washed three times with 1 mL of 50 mM Tris, 150 mM NaCl and 1% SDS, three times with 1% SDS in 8 M urea, and two times with PBS.To release proteins and enable analysis by SDS-PAGE, the beads were resuspended in 100 µL of Milli-Q water containing 5% formic acid to cleave the DADPS linker and incubated at room temperature for 2 h.The beads were pelleted at 2,000 xg for 1 min, and the supernatant was saved.The 5% formic acid cleavage step was repeated once.The beads were washed once with resuspension buffer and combined with the saved supernatants from both 5% formic acid treatments.The proteins were precipitated in chloroform/methanol/water, and equal amounts were separated using SDS-PAGE and analyzed using silver staining following the manufacturer protocol.To enable global identification and site mapping analysis of captured proteins by LC-MS/MS, they were subjected on-bead digestion followed by formic acid-mediated release of modified peptides as follows.
After subjecting ~1.5 mg biotinylated proteins to capture on streptavidin beads as described above, the beads were washed three times with 1 mL of 50 mM Tris, 150 mM NaCl and 1% SDS, three times with 1% SDS in 8 M urea, two times with PBS, and resuspended in 500 μL of 100 mM Tris containing 8 M urea.Next, tris(2-carboxyethyl)phosphine (TCEP) and chloroacetamide were added to final concentrations of 10 mM and 40 mM, respectively, and the beads were incubated at room temperature for 10 min.40 µg of mass spectrometry-grade lysyl endopepdtidase were added and incubated for 4 h at room temperature.100 mM Tris was added to bring the urea concentration to 2 mM, then 40 µg of sequencing grade modified trypsin was added and the beads were incubated overnight at room temperature with end-over-end rotation.
The beads were washed three times with PBS and three times with Milli-Q water and combined to form presumed unmodified peptides.The modified peptides were released from the beads with two sequential treatments of 200 µL of 5% formic acid.Next, the beads were washed once with 400 μL of 50% ACN/water and 1% formic acid and the wash was combined with the formic acid-released samples to generate modified peptide samples.The peptide samples were desalted using a 10 mg Strata X reversed-phase solid phase extraction cartridge, concentrated on a speed-vac, and analyzed by LC-MS/MS as described below.to allow for excitation of precursor ions within the quadrupole linear ion trap (i.e., activated ionelectron transfer dissociation (AI-ETD)). 15Modified peptide samples prepared as described above were separated using 120 min gradients on a 40 cm column packed in-house with C18 particles (BEH C18 1.7 µm, Waters) to 30,000 psi 16 held at 50 °C using a column heater.Mobile Data analysis.All files were searched using MSFragger (v.3.5) 18 via FragPipe interface (v.18.0).Spectra were searched against Corynebacterium glutamicum database downloaded from UniProt 2020101 and appended with a reverse decoy and common contaminant identifications.Default settings were used unless specifically stated.The custom variable modification with mass 251.1634 on S and T residues was added.Protease was set to "trypsin,"

Peptide-level enrichment (LC-MS/MS
and digest length was 6-65.PTMProphet 19 was enabled to localize ST:251.1634mod with min probability of 0.5.MaxLFQ quantification was enabled via IonQuant 20 with default settings, match-between-runs enabled, and MaxLFQ min ions set to 1. IPSA webtool 21 was used to annotate spectra and produce figures.A FragPipe search was also performed that allowed for the modification to occur on any residue other than Ser/Thr but there were no peptide matches. HCD and AI-ETD ion lists ____________ ** Supplementary figures ____________________________________________ S7 Figure S1.Efficiency of protein extraction method _______________________ S7 Figure S2.Comparison of O-AlkTMM and alkyne carboxylic acids __________ S8 Figure S3.Comparison of proteins identified in this study and predicted mycomembrane proteins _______________________________________ S8 Figure S4.Examples of annotated MS spectra _________________________ S9 Figure S5.AlphaFold structures of O-mycoloylated proteins _______________ S10 Experimental Methods ____________________________________________ S11 General procedures ______________________________________________ S11 Construction of mutants ___________________________________________ S13 Cmt1-dependent protein labeling ____________________________________ S14 Concentration-dependent protein labeling _____________________________ S15 Cell labeling and flow cytometry analysis ______________________________ S15 Click chemistry-mediated affinity enrichment using Az-TB _________________ S16 Label-free quantitative LC-MS/MS analysis (LC-MS/MS Study 1) ___________ S17 PorB labeling ___________________________________________________ S19 Click chemistry-mediated affinity enrichment using Az-DADPS-B ___________ S19 LC-MS/MS analysis and PTM site localization (LC-MS/MS Studies 2 and 3) ___ S22 SI References ____________________________________________________ S24 *LC-MS/MS raw and curated data available separately as "Supplementary Tables S4-11_Proteomic Analysis.xlsx"**Lists of ions observed in HCD and AI-ETD spectra in Figures 3E and 3F available separately as "Supplementary Tables S12-13_HCD and AI-ETD Ions.xlsx"

In a 96
well plate, 198 µL cultures of Cg MB001, MB001 Δcmt1, or MB001 Δcmt1::cmt1 in LB medium at an OD600 of 0.3 were treated with O-AlkTMM, 6-heptynoic acid, or 17octadecynoic acid at concentrations of 0.1 or 0.4 mM, or DMSO control, and incubated for 16 h.The cells were pelleted by centrifugation at 3,200 xg for 5 min, washed three times with PBS containing 0.5 mg/mL bovine serum albumin (BSA), and fixed in 4% formaldehyde in PBS for 10 min.The cell was pelleted and washed three times with PBS and resuspended in 138 µL of PBS.Next, the cells were treated with Az-488 (20 µM) under CuAAC conditions and incubated in the dark at room temperature for 30 min.The cells were pelleted and washed to remove excess unreacted reagents and resuspended in 200 µL of PBS. 5 µL of the resuspended cells were added to 200 µL of PBS and analyzed on a CytoFlex flow cytometer (Beckman Coulter).Mean were combined and concentrated to near dryness by rotary evaporation.Next, these proteins were precipitated by adding 40 mL of ether and keeping at -20 °C for 10 h.The precipitated proteins were pelleted by centrifugation, air-dried, and resuspended in 500 µL of Milli-Q water, and an equal amount of methanol was added.Then chloroform/methanol (2:1 v/v) was added to a total volume of 40 mL and the sample was kept at -20 °C overnight.The precipitated proteins were pelleted by centrifugation, air-dried, and dissolved in the resuspension buffer.Next, the cell pellet remaining following the chloroform/methanol extractions was resuspended in resuspension buffer and stirred at 60 °C for 1 h, then transferred to screw-cap vials containing 0.25 mL of 0.1 mm silica/zirconia beads and subjected to bead beating at 5.5 m/s for 20 s (repeated 3 times).The beads were allowed to settle then the supernatants were gently transferred into a 15 mL tube and centrifuged at 3,900 rpm for 5 min at room temperature.Next, the supernatant was gently transferred into another 15 mL tube and centrifuged at 10,000 xg for 5 min at room temperature.The clear supernatants were collected and combined with the proteins obtained by chloroform/methanol extraction.Next, ~1.5 mg of the combined protein extract was treated with Az-T-B (80 µM) under CuAAC conditions.Excess unreacted reagents were removed by chloroform/methanol/water precipitation as described above.The proteins were dried at 37 °C for 10 min and dissolved in 1 mL resuspension buffer.Protein-level enrichment.~100 µg of the 1.5 mg labeled protein were loaded onto 30 µL of settled pre-cleaned streptavidin agarose beads and incubated in the dark for 2 h at room temperature with end-over-end rotation.The beads were centrifuged at 2,000 xg for 1 min, and the supernatants were discarded.Next, the beads were washed twice with 50 mM Tris, 150 mM NaCl, and 1% SDS in water, twice with 1% SDS in 8 M urea, and twice with PBS.The bound proteins were eluted from the beads by boiling in 40 µL of 2x sample loading buffer at 95 °C for 15 min, separated using SDS-PAGE, and input and output protein sampls were analyzed by ingel fluorescence and silver staining.The remaining ~1.4 mg of Az-T-B labeled proteins were prepared for subsequent LC-MS/MS analysis.Proteins were enriched on 100 µL of streptavidin beads and eluted as described above, stacked in a polyacrylamide gel, and stained overnight with QC Colloidal Coomassie to prepare for in-gel digestion and LC-MS/MS analysis.

MS/MS Studies 2 and 3 )
Study 3).~1.5 mg of biotinylated protein extracts from probe-treated and untreated Cg wild type or Δcmt1 mutant prepared as described above were dissolved in 500 µL of 8 M urea in 100 mM Tris.TCEP and chloroacetamide were added to final concentrations of 10 mM and 40 mM, respectively, and incubated at room temperature for 10 min.100 mM Tris was added to bring the urea concentration to 2 mM, and 40 µg of sequence-grade modified trypsin was added and incubated overnight at RT with end-over-end rotation.Next, 40 µg of sequencing grade modified trypsin was added and incubated at room temperature for 4 h.The suspension was diluted with PBS to bring the urea concentration to 0.4 mM.Next, 100 µL of settled pre-cleaned streptavidin beads suspended in 200 µL of PBS were added and incubated for 4 h at room temperature with end-over-end rotation.The beads containing bound, modified peptides were pelleted by centrifugation at 2,000 xg for 3 min, and the supernatant was discarded.The beads were washed three times with PBS and three times with Milli-Q water.The enriched modified peptides were released from the beads with two sequential treatments of 200 µL of 5% formic acid.Next, the beads were washed once with 400 μL of 50% ACN/water and 1% formic acid and the wash was combined with the formic acidreleased samples to generate modified peptide samples.The modified peptide samples were desalted using a 10 mg Strata X reversed-phase solid phase extraction cartridge, concentrated on a speed-vac, and analyzed by LC-MS/MS as described below.LC-MS/MS analysis of modified peptides generated from Az-DADPS-B enrichment (LC-LC-MS/MS analysis.These MS experiments were performed on an Orbitrap FusionLumos Tribrid mass spectrometer (ThermoFisher Scientific) coupled to a nanoflow Ultimate 3000 Dionex pump (ThermoFisher Scientific).The mass spectrometer was retrofitted to include a Firestar ti60 Synrad 60 W CO2 continuous wave infrared (IR) laser (10.6 µm) (Mukilteo, WA) phase A (MPA) was 0.2% formic acid in H2O, mobile phase B (MPB) was 0.2% formic acid in 90:10 isopropanol:acetonitrile with 5 mM ammonium formate and 120 min methods ramped from 0-2% MPB over 5 min at 380 nL/min, 2-32% MPB over 67 min at 280 nL/min, 32-70% MPB over 8 min at 260 nL/min, 70-82% MPB over 5 min at 225 nL/min, 82-87% MPB over 12 min at 225 nL/min, 87-95% MPB over 1 min at 225 nL/min, 95-100% MPB over 8 min at 225 nL/min, held at 100% MPB for 3 min at 225 nL/min, 100-0% MPB over 1 min at 225 nL/min, and equilibrated at 0% MPB for 10 min at 225 nL/min. 17~500 ng peptides were injected per experiment.The spray voltage was +2.0 kV with respect to the ground.High-resolution MS1 and MS 2 scans were acquired in the positive ion mode in the Orbitrap at 60,000 and 15,000 resolution, respectively.MS 1 scans were collected every 2 seconds with m/z range of 300-1,650, AGC target 1e6, and a maximum injection time of 50 ms.Peaks were selected for MS/MS in a data-dependent manner, including precursors with charge states 2-6 and dynamic exclusion set to 15 sec.MS 2 scans were collected with m/z range of 150-1,650, AGC target 5e4, and a maximum injection time of 150 ms.For ion activation, either higher-energy C-trap dissociation (HCD) or activated ionelectron transfer dissociation (AI-ETD) was performed.Each method was performed in technical (injection) duplicates for each sample.HCD was performed at 28 NCE, and AI-ETD was performed with the laser set to 12% maximum power (~10 W).

Table S2 .
List of plasmids used in this study.

Table S3 .
List of primers used in this study.

Supplementary Figure S1. Efficiency of protein extraction method. Cg wild
type (WT) was cultured in the presence of 100 μM O-AlkTMM or left untreated for 16 h, then proteins were extracted successively three times, each by boiling at 100 °C in 4% SDS for 5 min (lanes 1-6: lanes 1&2, first extraction; lanes 3&4, second extraction; lanes 5&6, third extraction).The pellet remaining from these initial three extractions was subjected to three further successive extractions, each carried out by heating to 60 °C in PBS containing 2 mg/mL lysozyme for 2 h, bead beating, and extraction with 2% SDS at 60 °C for 2 h (lanes 7-12: lanes 7&8, fourth extraction; lanes 9&10, fifth extraction; lanes 11&12, sixth extraction).The proteins from each extraction step were subjected to CuAAC with azido-488, separated by SDS-PAGE, and visualized by Coomassie Brilliant Blue (CBB) staining and in-gel fluorescence scanning.
13tegration of pK-PIM derivative plasmids13was validated by colony PCR.chloroform/methanol/water precipitation.The proteins were air-dried briefly and dissolved in resuspension buffer, then equal amounts of each sample were resolved using SDS-PAGE and analyzed using in-gel fluorescence scanning and Coomassie staining.
Briefly, the pCRD206 derivative containing homology regions of the gene of interest was electroporated into wild-type MB001 C. glutamicum.To recover transformants, cells were plated to BHI + kanamycin at 24C to allow for plasmid replication.Transformants were restreaked to BHI + kanamycin at 30C to isolate plasmid integrants.Integrants were plated to BHI + 10% sucrose at 30C to select against sacB encoded on the pCRD206 vector.Candidate mutants were further screened by replica patching to BHI and BHI + kanamycin to identify kanamycin-sensitive colonies.Deletion mutants were confirmed by performing colony PCR using primers that anneal upstream and downstream to the gene of interest.chloroform/methanol/water, and resuspended in resuspension buffer.The resuspended proteins were reacted with Az-488 (20 µM) under CuAAC conditions, and excess unreacted reagents were removed using