title: Glycan side reaction may compromise ETD-based glycopeptide identification.
abstract: Tris(hydroxymethyl)aminomethane (Tris) is one of the most frequently used buffer ingredients. Among other things, it is recommended and is usually used for lectin-based affinity enrichment of glycopeptides. Here we report that sialic acid, a common 'capping' unit in both N- and O-linked glycans may react with this chemical, and this side reaction may compromise glycopeptide identification when ETD spectra are the only MS/MS data used in the database search. We show that the modification may alter N- as well as O-linked glycans, the Tris-derivative is still prone to fragmentation both in 'beam-type' CID (HCD) and ETD experiments, at the same time--since the acidic carboxyl group was 'neutralized'--it will display a different retention time than its unmodified counterpart. We also suggest solutions that--when incorporated into existing search engines--may significantly improve the reliability of glycopeptide assignments.
authors.affiliation: Laboratory of Proteomics Research, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary, darula.zsuzsanna@brc.mta.hu.
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Darula
authors.forename: Katalin F
authors.initials: KF
authors.lastname: Medzihradszky
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D006020:Glycopeptides / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D007477:Ions / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods / Q000592:standards*; D015203:Reproducibility of Results; D013574:Synaptosomes / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods / Q000592:standards*
chemical_list: D006020:Glycopeptides; D007477:Ions; D010446:Peptide Fragments; D019158:N-Acetylneuraminic Acid