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J Proteome Res. 2019 Feb 1;18(2):652-663. doi: 10.1021/acs.jproteome.8b00638. Epub 2018 Dec 21.

A Tandem Mass Spectrometry Sequence Database Search Method for Identification of O-Fucosylated Proteins by Mass Spectrometry.

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Institute for Systems Biology , Seattle , Washington 98109 , United States.
Proteomics Resource , University of Washington , Seattle , Washington 98195 , United States.
Center for Global Infectious Disease Research , Seattle Children's Research Institute , Seattle , Washington 98101 , United States.
Harvard Medical School and Boston Children's Hospital , Boston , Massachusetts 02115 , United States.


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.


C-mannosylation; O-fucosylation; Plasmodium; thrombospondin type 1 repeat

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