title: Identification of N-linked glycosylation sites using glycoprotein digestion with pronase prior to MALDI tandem time-of-flight mass spectrometry.
abstract: Glycopeptides are typically prepared by cleaving the proteins with specific proteolytic enzymes, such as trypsin. The resulting glycopeptides tend to have weak mass spectrometry ion signals (ESI or MALDI) due to their relatively large molecular weight. The identification of glycosylation sites with tandem mass spectrometry is further complicated by fragmentation of both the peptide backbone and the glycan moiety. We explored a method using a nonspecific enzyme, pronase, to generate small glycopeptides (between two and six amino acids). These glycopeptides were enriched and desalted using a microscale hydrophilic interaction chromatography extraction device prior to MALDI QTof MS analysis. MALDI matrix, 2, 5-dihydroxybenzoic acid, doped with ammonium triscitrate, was utilized for analysis. Sodiated ions were observed as minor ions, while protonated ions were enhanced dramatically with this matrix. Collision-induced dissociation was performed on both the protonated and sodiated ions. MS/MS fragmentation spectra reveal that proton has greater affinity for the peptide moiety, while the sodium cation tends to associate with the sugar moiety. Characteristic fragment patterns allowed for identifications of glycosylation sites for both the protonated and the sodiated precursor ions. Model proteins, horseradish peroxidase and alpha1-acid glycoproteins, were analyzed to illustrate the identification of N-linked glycosylation sites and data interpretation algorithm.
authors.affiliation: Life Sciences R and D, Waters Corporation, Milford, Massachusetts 01757, USA. ying_qing_yu@waters.com
authors.forename: Ying Qing
authors.initials: YQ
authors.lastname: Yu
authors.forename: Jennifer
authors.initials: J
authors.lastname: Fournier
authors.forename: Martin
authors.initials: M
authors.lastname: Gilar
authors.forename: John C
authors.initials: JC
authors.lastname: Gebler
mesh_terms: D006023:Glycoproteins / Q000032:analysis*; D006031:Glycosylation; D015394:Molecular Structure; D011402:Pronase / Q000737:chemistry*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D006023:Glycoproteins; D011402:Pronase