posted on 2018-12-07, 00:00authored byKristian E. Swearingen, Jimmy K. Eng, David Shteynberg, Vladimir Vigdorovich, Timothy A. Springer, Luis Mendoza, D. Noah Sather, Eric W. Deutsch, Stefan H. I. Kappe, Robert L. Moritz
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.