title: Semi-automated identification of N-Glycopeptides by hydrophilic interaction chromatography, nano-reverse-phase LC-MS/MS, and glycan database search.
abstract: Glycoproteins fulfill many indispensable biological functions, and changes in protein glycosylation have been observed in various diseases. Improved analytical methods are needed to allow a complete characterization of this complex and common post-translational modification. In this study, we present a workflow for the analysis of the microheterogeneity of N-glycoproteins that couples hydrophilic interaction and nanoreverse-phase C18 chromatography to tandem QTOF mass spectrometric analysis. A glycan database search program, GlycoPeptideSearch, was developed to match N-glycopeptide MS/MS spectra with the glycopeptides comprised of a glycan drawn from the GlycomeDB glycan structure database and a peptide from a user-specified set of potentially glycosylated peptides. Application of the workflow to human haptoglobin and hemopexin, two microheterogeneous N-glycoproteins, identified a total of 57 distinct site-specific glycoforms in the case of haptoglobin and 14 site-specific glycoforms of hemopexin. Using glycan oxonium ions and peptide-characteristic glycopeptide fragment ions and by collapsing topologically redundant glycans, the search software was able to make unique N-glycopeptide assignments for 51% of assigned spectra, with the remaining assignments primarily representing isobaric topological rearrangements. The optimized workflow, coupled with GlycoPeptideSearch, is expected to make high-throughput semiautomated glycopeptide identification feasible for a wide range of users.
authors.affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University , 3970 Reservoir Road NW, Washington, DC 20057-1465, United States.
authors.forename: Petr
authors.initials: P
authors.lastname: Pompach
authors.forename: Kevin B
authors.initials: KB
authors.lastname: Chandler
authors.forename: Renny
authors.initials: R
authors.lastname: Lan
authors.forename: Nathan
authors.initials: N
authors.lastname: Edwards
authors.forename: Radoslav
authors.initials: R
authors.lastname: Goldman
mesh_terms: D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D030562:Databases, Protein; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D006242:Haptoglobins / Q000737:chemistry / Q000302:isolation & purification; D006466:Hemopexin / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping / Q000379:methods; D011134:Polysaccharides / Q000737:chemistry / Q000302:isolation & purification*; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification; D059748:Proteolysis; D012984:Software; D053719:Tandem Mass Spectrometry
chemical_list: D006023:Glycoproteins; D006242:Haptoglobins; D010446:Peptide Fragments; D011134:Polysaccharides; D020033:Protein Isoforms; D006466:Hemopexin