Quantitative proteome-wide O-glycoproteomics analysis with FragPipe

Abstract

Identification of O-glycopeptides from tandem mass spectrometry data is complicated by the near complete dissociation of O-glycans from the peptide during collisional activation and by the combinatorial explosion of possible glycoforms when glycans are retained intact in electron-based activation. The recent O-Pair search method provides an elegant solution to these problems, using a collisional activation scan to identify the peptide sequence and total glycan mass, and a follow-up electron-based activation scan to localize the glycosite(s) using a graph-based algorithm in a reduced search space. Our previous O-glycoproteomics methods with MSFragger-Glyco allowed for extremely fast and sensitive identification of O-glycopeptides from collisional activation data but had limited support for site localization of glycans and quantification of glycopeptides. Here, we report an improved pipeline for O-glycoproteomics analysis that provides proteome-wide, site-specific, quantitative results by incorporating the O-Pair method as a module within FragPipe. In addition to improved search speed and sensitivity, we add flexible options for oxonium ion-based filtering of glycans and support for a variety of MS acquisition methods and provide a comparison between all software tools currently capable of O-glycosite localization in proteome-wide searches.

Keywords: Glycoproteomics; Localization; O-Glycopeptides; Software.

Fig. 1

Workflow of O-Pair in FragPipe. Paired MS2 scans of a glycopeptide are analyzed using MSFragger-Glyco search to identify the peptide and total glycan mass from the HCD scan. EthcD-only data can also be searched as an alternative option. O-Pair deconvolutes the total glycan mass to individual localized glycans using the EThcD scan only. O-Pair results are written back to the FragPipe results tables for quantitation

Fig. 2

Comparison of 4-protein mixture O-glycoPSMs. A Total gPSMs identified by each software tool. *Byonic results are taken from Lu et al. [20] and allow a maximum of 3 glycans per peptide. All other tools allow a maximum of 5 glycans. B Comparison of FragPipe and MetaMorpheus O-Pair gPSMs by localization confidence level, indicating that the increase in FragPipe gPSMs is driven by Level 3 gPSMs

Fig. 3

Comparison of urine O-glycoproteomics results by localization confidence level

Fig. 4

Comparison of human skin cell line O-glycoproteomics results. A gPSMs identified by confidence level for each software tool in one LC–MS file, using a database of 32 glycans. B Glyco PSMs, peptides, sites, and proteins identified by FragPipe O-Pair search in all 8 fractions of 4 knockouts (32 LC–MS files total). C Example volcano plot of quantified glycosites from GALNT-6 knockout data. Detected GALNT6 glycosites are highlighted as an example, showing large reductions in abundance following knockout

Fig. 5

Entrapment protein and glycan searches. A Total gPSMs identified for searches of human skin cell line data using human-only (blue bars) or human plus A. thaliana protein databases (gray bars). Pie charts indicate the numbers of human (blue) or A. thaliana (yellow) gPSMs reported at 1% FDR. B Glycan entrapment search of IMHO-treated cell lines. Hexose-containing glycans are expected only at low levels due to IMHO treatment. Bars show gPSMs identified after oxonium ion filtering (FragPipe) or at 1% glycan FDR (pGlyco3). Pie charts show the proportion of gPSMs with hexose (orange) and non-hexose (blue) containing glycan(s) for each search