Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Feb;417(5):921-930.
doi: 10.1007/s00216-024-05382-x. Epub 2024 Jun 15.

Quantitative proteome-wide O-glycoproteomics analysis with FragPipe

Daniel A Polasky  1 , Lei Lu  2   3 , Fengchao Yu  4 , Kai Li  5 , Michael R Shortreed  2 , Lloyd M Smith  2 , Alexey I Nesvizhskii  6   7
Affiliations

Quantitative proteome-wide O-glycoproteomics analysis with FragPipe

Daniel A Polasky et al. Anal Bioanal Chem. 2025 Feb.

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.

PubMed Disclaimer

Conflict of interest statement

Declarations. Conflict of interest: D. A. P., F. Y., and A. I. N. receive royalties from the University of Michigan for the sale of MSFragger and IonQuant software licenses to commercial entities. All license transactions are managed by the University of Michigan Innovation Partnerships office, and all proceeds are subject to university technology transfer policy. The other authors have no competing interests to declare.

Figures

Fig. 1
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
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
Fig. 3
Comparison of urine O-glycoproteomics results by localization confidence level
Fig. 4
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
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
See this image and copyright information in PMC

References

    1. Varki A. Biological roles of glycans. Glycobiology. 2017;27(1):3–49. - DOI - PMC - PubMed
    1. Bagdonaite I, Malaker SA, Polasky DA, Riley NM, Schjoldager K, Vakhrushev SY, et al. Glycoproteomics. Nat Rev Methods Prim. 2022;2(1):48-. - DOI
    1. Thaysen-Andersen M, Packer NH, Schulz BL. Maturing glycoproteomics technologies provide unique structural insights into the n-glycoproteome and its regulation in health and Disease. Mol Cell Proteomics. 2016;15(6):1773–90. - DOI - PMC - PubMed
    1. Wisnovsky S, Bertozzi CR. Reading the glyco-code: new approaches to studying protein-carbohydrate interactions. Curr Opin Struct Biol. 2022;75:102395. - DOI - PMC - PubMed
    1. RodrIguez E, Schetters STT, van Kooyk Y. The tumour glyco-code as a novel immune checkpoint for immunotherapy. Nat Rev Immunol. 2018;18(3):204–11. - DOI - PubMed

LinkOut - more resources