Glycan–protein cross-linking mass spectrometry reveals sialic acid-mediated protein networks on cell surfaces †

A cross-linking method is developed to elucidate glycan-mediated interactions between membrane proteins through sialic acids. The method provides information on previously unknown extensive glycomic interactions on cell membranes. The vast majority of membrane proteins are glycosylated with complicated glycan structures attached to the polypeptide backbone. Glycan–protein interactions are fundamental elements in many cellular events. Although significant advances have been made to identify protein–protein interactions in living cells, only modest advances have been made on glycan–protein interactions. Mechanistic elucidation of glycan–protein interactions has thus far remained elusive. Therefore, we developed a cross-linking mass spectrometry (XL-MS) workflow to directly identify glycan–protein interactions on the cell membrane using liquid chromatography-mass spectrometry (LC-MS). This method involved incorporating azido groups on cell surface glycans through biosynthetic pathways, followed by treatment of cell cultures with a synthesized reagent, N-hydroxysuccinimide (NHS)–cyclooctyne, which allowed the cross-linking of the sialic acid azides on glycans with primary amines on polypeptide backbones. The coupled peptide–glycan–peptide pairs after cross-linking were identified using the latest techniques in glycoproteomic and glycomic analyses and bioinformatics software. With this approach, information on the site of glycosylation, the glycoform, the source protein, and the target protein of the cross-linked pair were obtained. Glycoprotein–protein interactions involving unique glycoforms on the PNT2 cell surface were identified using the optimized and validated method. We built the GPX network of the PNT2 cell line and further investigated the biological roles of different glycan structures within protein complexes. Furthermore, we were able to build glycoprotein–protein complex models for previously unexplored interactions. The method will advance our future understanding of the roles of glycans in protein complexes on the cell surface.

The cross-linking reactions between glycans and proteins depended greatly on the efficiency of the ManNAz incorporation in the cell.To monitor the incorporation we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) and profiled the resulting N-glycans using methods described previously. 2 The N-glycomic profile yielded over 300 sialylated N-glycans in the PNT2 cell line (Figure S12).The SiaNAz-modified glycans were readily identified based on their corresponding masses.Tandem MS was used to confirm the incorporation using diagnostic peaks corresponding to the SiaNAz cation (m/z = 333.10).To obtain maximum incorporation, we further monitored the expression of SiaNAz-containing N-glycans with regard to treatment time.As shown in Figure S1a and b, up to 90% of the total sialic acid could be converted to SiaNAz after 72 hours of ManNAz treatment.

Validation of cross-linking reaction on cell lines
To determine first the extent of the cross-linking reactions, we employed gel electrophoresis to analyze the products.The proteins were digested to peptides, and the molecular weight ranged from 3k Da to 5kDa with the cross-linker while the masses of around 2k Da were observed without the cross-linker modification (control) (Figure S13a).

Validation of enrichment method
With methods for analyzing the crosslink products, we optimized enrichment methods for the resulting GPX pairs.PPX products were commonly fractionated using strong cation exchange (SCX) or size exclusion chromatography (SEC), while glycopeptides were usually separated from peptide background using hydrophilic interaction chromatography (HILIC). 3,4We tested the enrichment of GPX products using SCX cartridge and HILIC.As shown in Figure S13b, around 200 GPX pairs were identified using SCX, while only 30 GPX pairs were identified using the combination of SCX and HILIC.These results suggested that one-step SCX is sufficient for the enrichment of GPX products prior to MS analysis.

Validation of GPX analysis workflow
We further validated the workflow with lectins that are known to bind sialic acid.
With the cross-linker in place, Sambucus nigra agglutinin (SNA) was reacted with the cell membrane containing SiaNAz (Figure S14a).From the LC-MS/MS data, the resulting GPX pairs included membrane glycopeptides with an SNA peptide.More than 100 of GPX products containing SNA peptides were identified, which corresponded to approximately 50 unique glycoprotein-SNA pairs.We compared the glycoproteins crosslinked by SNA to those previously identified as potentially SNA-binding proteins using a proximity approach (Lectin PROXL).In an earlier study, SNA was modified with Fe 3+ probe to oxidize proteins that were in the proximity of SNA. 5 As shown in Figure S14b, a large fraction (60%) of SNA-cross-linked glycoproteins was also found by our previously developed method further validating the results of the cross-linking method.
SiaNAz-containing glycans in PNT2 cells.Different glycans have variable incorporation rate, while over 80% was achieved on average.(c) Tandem MS of released N-glycans from cell membrane after click addition of a crosslinker.The MS/MS spectra validate the composition and provide structural information regarding the reacted glycan.Figure S7 GPX analysis indicated that the glycan composition having core fucose interacted more with other proteins (inter) than itself (intra).Conversely, the absence of the fucose resulted in significantly more interact with the parent protein (intra).

Figure S10
The synthetic route for the synthesis of the cross-linker, NHS-cyclooctyne.
Around 40% total yield was achieved after four steps.

Figure S2 Figure S3
Figure S2 Representative identified tandem mass spectrums of cross-linked GPX pairs

Figure S4 1 Interaction Network (Unidentified) 2 Figure
Figure S4Target proteins captured by sialylated proteins were found to overlap with the

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Figure S6 (a) The biological function of source (left) and target (right) proteins.(b) The

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Figure S9 PNT2 migration was accelerated after cell surface sialic acid was lost.(a)

Figure S12
Figure S12 LC-MS profile of N-Glycans released from PNT2 cells (top).Annotated

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Figure S13 (a) SDS-PAGE results of digested samples from control (no cross-linker) and

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Figure S14 (a) Schematic diagram of the SNA-glycoprotein cross-linking on the cell