title: Exploring site-specific N-glycosylation microheterogeneity of haptoglobin using glycopeptide CID tandem mass spectra and glycan database search.
abstract: Glycosylation is a common protein modification with a significant role in many vital cellular processes and human diseases, making the characterization of protein-attached glycan structures important for understanding cell biology and disease processes. Direct analysis of protein N-glycosylation by tandem mass spectrometry of glycopeptides promises site-specific elucidation of N-glycan microheterogeneity, something that detached N-glycan and deglycosylated peptide analyses cannot provide. However, successful implementation of direct N-glycopeptide analysis by tandem mass spectrometry remains a challenge. In this work, we consider algorithmic techniques for the analysis of LC-MS/MS data acquired from glycopeptide-enriched fractions of enzymatic digests of purified proteins. We implement a computational strategy that takes advantage of the properties of CID fragmentation spectra of N-glycopeptides, matching the MS/MS spectra to peptide-glycan pairs from protein sequences and glycan structure databases. Significantly, we also propose a novel false discovery rate estimation technique to estimate and manage the number of false identifications. We use a human glycoprotein standard, haptoglobin, digested with trypsin and GluC, enriched for glycopeptides using HILIC chromatography, and analyzed by LC-MS/MS to demonstrate our algorithmic strategy and evaluate its performance. Our software, GlycoPeptideSearch (GPS), assigned glycopeptide identifications to 246 of the spectra at a false discovery rate of 5.58%, identifying 42 distinct haptoglobin peptide-glycan pairs at each of the four haptoglobin N-linked glycosylation sites. We further demonstrate the effectiveness of this approach by analyzing plasma-derived haptoglobin, identifying 136 N-linked glycopeptide spectra at a false discovery rate of 0.4%, representing 15 distinct glycopeptides on at least three of the four N-linked glycosylation sites. The software, GlycoPeptideSearch, is available for download from http://edwardslab.bmcb.georgetown.edu/GPS .
authors.affiliation: Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC 20007, USA.
authors.forename: Kevin Brown
authors.initials: KB
authors.lastname: Chandler
authors.forename: Petr
authors.initials: P
authors.lastname: Pompach
authors.forename: Radoslav
authors.initials: R
authors.lastname: Goldman
authors.forename: Nathan
authors.initials: N
authors.lastname: Edwards
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D016477:Artifacts; D002853:Chromatography, Liquid; D016208:Databases, Factual; D006031:Glycosylation; D006242:Haptoglobins / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis*; D011134:Polysaccharides / Q000032:analysis*; D059748:Proteolysis; D012984:Software; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
chemical_list: D006242:Haptoglobins; D010446:Peptide Fragments; D011134:Polysaccharides; D014357:Trypsin