title: Site-specific N-glycosylation analysis: matrix-assisted laser desorption/ionization quadrupole-quadrupole time-of-flight tandem mass spectral signatures for recognition and identification of glycopeptides.
abstract: The identification of glycosylation sites in proteins is often possible through a combination of proteolytic digestion, separation, mass spectrometry (MS) and tandem MS (MS/MS). Liquid chromatography (LC) in combination with MS/MS has been a reliable method for detecting glycopeptides in digestion mixtures, and for assigning glycosylation sites and glycopeptide sequences. Direct interfacing of LC with MS relies on electrospray ionization, which produces ions with two, three or four charges for most proteolytic peptides and glycopeptides. MS/MS spectra of such glycopeptide ions often lead to ambiguous interpretation if deconvolution to the singly charged level is not used. In contrast, the matrix-assisted laser desorption/ionization (MALDI) technique usually produces singly charged peptide and glycopeptide ions. These ions require an extended m/z range, as provided by the quadrupole-quadrupole time-of-flight (QqTOF) instrument used in these experiments, but the main advantages of studying singly charged ions are the simplicity and consistency of the MS/MS spectra. A first aim of the present study is to develop methods to recognize and use glycopeptide [M+H]+ ions as precursors for MS/MS, and thus for glycopeptide/glycoprotein identification as part of wider proteomics studies. Secondly, this article aims at demonstrating the usefulness of MALDI-MS/MS spectra of N-glycopeptides. Mixtures of diverse types of proteins, obtained commercially, were prepared and subjected to reduction, alkylation and tryptic digestion. Micro-column reversed-phase separation allowed deposition of several fractions on MALDI plates, followed by MS and MS/MS analysis of all peptides. Glycopeptide fractions were identified after MS by their specific m/z spacing patterns (162, 203, 291 u) between glycoforms, and then analyzed by MS/MS. In most cases, MS/MS spectra of [M+H]+ ions of glycopeptides featured peaks useful for determining sugar composition, peptide sequence, and thus probable glycosylation site. Peptide-related product ions could be used in database search procedures and allowed the identification of the glycoproteins.
authors.affiliation: Manitoba Centre for Proteomics, University of Manitoba, Winnipeg, Manitoba, Canada. krokhino@cc.umanitoba.ca
authors.forename: Oleg
authors.initials: O
authors.lastname: Krokhin
authors.forename: Werner
authors.initials: W
authors.lastname: Ens
authors.forename: Kenneth G
authors.initials: KG
authors.lastname: Standing
authors.forename: John
authors.initials: J
authors.lastname: Wilkins
authors.forename: Hélène
authors.initials: H
authors.lastname: Perreault
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D045424:Complex Mixtures / Q000032:analysis / Q000737:chemistry; D005240:Feasibility Studies; D017127:Glycation End Products, Advanced / Q000032:analysis* / Q000737:chemistry*; D006020:Glycopeptides / Q000032:analysis* / Q000737:chemistry*; D006031:Glycosylation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D011485:Protein Binding; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013057:Spectrum Analysis / Q000379:methods
chemical_list: D045424:Complex Mixtures; D017127:Glycation End Products, Advanced; D006020:Glycopeptides; D010446:Peptide Fragments