Journal of proteome research
Databases, Protein; Fucose; Glycosylation; Peptides; Plasmodium; Proteomics; Search Engine; Tandem Mass Spectrometry
Thrombospondin type 1 repeats (TSRs), small adhesive protein domains with a wide range of functions, are usually modified with O-linked fucose, which may be extended to O-fucose-β1,3-glucose. Collision-induced dissociation (CID) spectra of O-fucosylated peptides cannot be sequenced by standard tandem mass spectrometry (MS/MS) sequence database search engines because O-linked glycans are highly labile in the gas phase and are effectively absent from the CID peptide fragment spectra, resulting in a large mass error. Electron transfer dissociation (ETD) preserves O-linked glycans on peptide fragments, but only a subset of tryptic peptides with low m/ z can be reliably sequenced from ETD spectra compared to CID. Accordingly, studies to date that have used MS to identify O-fucosylated TSRs have required manual interpretation of CID mass spectra even when ETD was also employed. In order to facilitate high-throughput, automatic identification of O-fucosylated peptides from CID spectra, we re-engineered the MS/MS sequence database search engine Comet and the MS data analysis suite Trans-Proteomic Pipeline to enable automated sequencing of peptides exhibiting the neutral losses characteristic of labile O-linked glycans. We used our approach to reanalyze published proteomics data from Plasmodium parasites and identified multiple glycoforms of TSR-containing proteins.
Institute for Systems Biology
Swearingen, Kristian E; Eng, Jimmy K; Shteynberg, David; Vigdorovich, Vladimir; Springer, Timothy A; Mendoza, Luis; Sather, D Noah; Deutsch, Eric W; Kappe, Stefan H I; and Moritz, Robert L, "A Tandem Mass Spectrometry Sequence Database Search Method for Identification of O-Fucosylated Proteins by Mass Spectrometry." (2019). Articles, Abstracts, and Reports. 2791.