A Classifier Based on Accurate Mass Measurements to Aid Large Scale, Unbiased Glycoproteomics

Abstract

Determining which glycan moieties occupy specific N-glycosylation sites is a highly challenging analytical task. Arguably, the most common approach involves LC-MS and LC-MS/MS analysis of glycopeptides generated by proteases with high cleavage site specificity; however, the depth achieved by this approach is modest. Nonglycosylated peptides are a major challenge to glycoproteomics, as they are preferentially selected for data-dependent MS/MS due to higher ionization efficiencies and higher stoichiometric levels in moderately complex samples. With the goal of improving glycopeptide coverage, a mass defect classifier was developed that discriminates between peptides and glycopeptides in complex mixtures based on accurate mass measurements of precursor peaks. By using the classifier, glycopeptides that were not fragmented in an initial data-dependent acquisition run may be targeted in a subsequent analysis without any prior knowledge of the glycan or protein species present in the mixture. Additionally, from probable glycopeptides that were poorly fragmented, tandem mass spectra may be reacquired using optimal glycopeptide settings. We demonstrate high sensitivity (0.892) and specificity (0.947) based on an in silico dataset spanning >100,000 tryptic entries. Comparable results were obtained using chymotryptic species. Further validation using published data and a fractionated tryptic digest of human urinary proteins was performed, yielding a sensitivity of 0.90 and a specificity of 0.93. Lists of glycopeptides may be generated from an initial proteomics experiment, and we show they may be efficiently targeted using the classifier. Considering the growing availability of high accuracy mass analyzers, this approach represents a simple and broadly applicable means of increasing the depth of MS/MS-based glycoproteomic analyses. Molecular &

A Comparison of Energy-Resolved Vibrational Activation/Dissociation Characteristics of Protonated and Sodiated High Mannose N-Glycopeptides

“…In particular, the analysis of protein glycosylation at the level of proteolytic glycopeptides is of high interest because, unlike methods which involve glycan release, this approach confers protein site specificity to analysis of the modifying glycans [5][6][7][8]. While advantageous in allowing for glycans of specific compositions and topologies to be localized to specific sites of a glycoprotein, these means of glycoprotein characterization can also be technically challenging since the heterogeneities and structural complexities of the oligosaccharide and the polypeptide must be addressed simultaneously [9][10][11]. In this respect, tandem mass spectrometry (MS/MS) methods are of central importance in interrogating the compositions and overall connectivities of glycopeptides [12,13].…”

Energy-resolved collision-induced dissociation pathways of model N-linked glycopeptides: implications for capturing glycan connectivity and peptide sequence in a single experiment

“…[19][20][21] While advantageous in terms of directness (glycan release is avoided, thus attachment sites need not be merely inferred), this approach is subject to a number of analytical challenges. 22,23 The acquisition and subsequent interpretation of informative tandem mass spectrometry (MS/MS) data for glycopeptide ions stand among the most pressing of these challenges. Signicant effort has been made to maximize the information content of glycopeptide ion dissociation spectra, including those obtained through vibrational activation/dissociation methods such as collisioninduced dissociation (CID) 24,25 and infrared multiphoton dissociation (IRMPD); 26,27 ion-electron and ion-ion reactions resulting in electron capture dissociation (ECD) 28,29 or electron transfer dissociation (ETD); 30,31 and irradiation with ultraviolet photons in order to achieve ultraviolet photodissociation (UVPD).…”

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

“…The original product-dependent (PD) method, which triggered ETD events upon recognition of m/z 204.086 in HCD-MS/MS (125), was lately followed up with a more informative approach where alternating ion trap CID and ETD events are triggered upon oxonium ion recognition in HCD-MS/MS spectra (126). In another innovative LC-MS/MS study, N-glycopeptides in nonenriched peptide mixtures were crudely isolated (postacquisition) from their nonglycosylated counterparts based on their accurate mass (127). This type of glycopeptide classification was based on a common mass defect of N-glycopeptides arising from their oxygen-rich elemental compositions.…”