Glycopeptide Analysis, Recent Developments and Applications

Desaire, Heather

doi:10.1074/mcp.r112.026567

Cited by 93 publications

(86 citation statements)

References 48 publications

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“…Furthermore, the BRB glycopeptide exhibited some examples of peptide fragmentation without complete loss of the glycan. For instance, the 0,2 X 1 + b 5 and Y 1 + b 5 internal fragments were among the most abundant signals in the DU ¼ 55.0 V CID spectrum (Fig. 3c).…”

Section: Analyst Papermentioning

confidence: 94%

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

Kolli

Dodds

2014

Analyst

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"Energy-resolved collision-induced dissociation pathways of model N-linked glycopeptides: implications for capturing glycan connectivity and peptide sequence in a single experiment" (2014). Faculty Publications --Chemistry Department. 68. http://digitalcommons.unl.edu/chemfacpub/68Energy-resolved collision-induced dissociation pathways of model N-linked glycopeptides: implications for capturing glycan connectivity and peptide sequence in a single experiment † Venkata Kolli and Eric D. Dodds * Tandem mass spectrometry (MS/MS) of glycopeptides stands among the principal analytical approaches for assessing protein glycosylation in a site-specific manner. The aims of such experiments are often to determine the monosaccharide connectivity of the glycan, the amino acid sequence of the peptide, and the site of glycan attachment. This level of detail is often difficult to achieve using any single ion dissociation method; however, precedent does exist for use of collision-induced dissociation (CID) to establish either the connectivity of the oligosaccharide or the sequence of the polypeptide depending upon the applied collision energy. Unfortunately, the relative energy requirements for glycan and peptide cleavage have not been thoroughly characterized with respect to specific physicochemical characteristics of the precursor ions. This report describes case studies on the energy-resolved CID pathways of model tryptic glycopeptides derived from Erythrina cristagalli lectin and bovine ribonuclease B. While glycopeptide ions having disparate physical and chemical characteristics shared strikingly similar qualitative responses to increasing vibrational energy deposition, the absolute collision energies at which either glycan or peptide fragmentations were accessed varied substantially among the precursor ions examined. Nevertheless, these data suggest that the energy requirements for peptide and glycan cleavage may be somewhat predictable based on characteristics of the precursor ion. The practical usefulness of these observations was demonstrated through implementation of online collision energy modulation such that both glycan and peptide fragmentation were captured in the same spectrum, providing near-exhaustive glycopeptide characterization in a single experiment. Overall, these results highlight the potential to further extend the capabilities of CID in the context of glycoproteomics.

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Section: Analyst Papermentioning

confidence: 94%

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

Kolli

Dodds

2014

Analyst

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“…Advances in glycopeptide identification have certainly been made through the utilization of alternative MS/MS techniques such as electron transfer dissociation (ETD) 14–17 and ultraviolet photodissociation (UVPD) 18,19 to improve the fragmentation information generated through MS/MS. Furthermore, data-independent analysis (DIA) of glycopeptides has recently been applied to glycopeptide analysis to circumvent the traditional reliance on data-dependent acquisition (DDA) of LC-MS/MS data, 20–22 which relies on peak picking of the most abundant species from the MS1 spectra to initiate MS/MS, a process that can yield incomplete site-specific characterization.…”

Section: Introductionmentioning

confidence: 99%

Data-independent oxonium ion profiling of multi-glycosylated biotherapeutics

Madsen

Farutin

Lin

et al. 2018

mAbs

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The characterization of glycosylation is required for many protein therapeutics. The emergence of antibody and antibody-like molecules with multiple glycan attachment sites has rendered glycan analysis increasingly more complicated. Reliance on site-specific glycopeptide analysis is therefore necessary to fully analyze multi-glycosylated biotherapeutics. Established glycopeptide methodologies have generally utilized a priori knowledge of the glycosylation states of the investigated protein(s), database searching of results generated from data-dependent liquid chromatography–tandem mass spectrometry workflows, and extracted ion quantitation of the individual identified species. However, the inherent complexity of glycosylation makes predicting all glycoforms on all glycosylation sites extremely challenging, if not impossible. That is, only the “knowns” are assessed. Here, we describe an agnostic methodology to qualitatively and quantitatively assess both “known” and “unknown” site-specific glycosylation for biotherapeutics that contain multiple glycosylation sites. The workflow uses data-independent, all ion fragmentation to generate glycan oxonium ions, which are then extracted across the entirety of the chromatographic timeline to produce a glycan-specific “fingerprint” of the glycoprotein sample. We utilized both HexNAc and sialic acid oxonium ion profiles to quickly assess the presence of Fab glycosylation in a therapeutic monoclonal antibody, as well as for high-throughput comparisons of multi-glycosylated protein drugs derived from different clones to a reference product. An automated method was created to rapidly assess oxonium profiles between samples, and to provide a quantitative assessment of similarity.

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“…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].…”

Section: Introductionmentioning

confidence: 99%

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

Aboufazeli

Kolli

Dodds

2015

J. Am. Soc. Mass Spectrom.

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Abstract. Fragmentation of glycopeptides in tandem mass spectrometry (MS/MS) plays a pivotal role in site-specific protein glycosylation profiling by allowing specific oligosaccharide compositions and connectivities to be associated with specific loci on the corresponding protein. Although MS/MS analysis of glycopeptides has been successfully performed using a number of distinct ion dissociation methods, relatively little is known regarding the fragmentation characteristics of glycopeptide ions with various charge carriers. In this study, energy-resolved vibrational activation/ dissociation was examined via collision-induced dissociation for a group of related high mannose tryptic glycopeptides as their doubly protonated, doubly sodiated, and hybrid protonated sodium adduct ions. The doubly protonated glycopeptide ions with various compositions were found to undergo fragmentation over a relatively low but wide range of collision energies compared with the doubly sodiated and hybrid charged ions, and were found to yield both glycan and peptide fragmentation depending on the applied collision energy. By contrast, the various doubly sodiated glycopeptides were found to dissociate over a significantly higher but narrow range of collision energies, and exhibited only glycan cleavages. Interestingly, the hybrid protonated sodium adduct ions were consistently the most stable of the precursor ions studied, and provided fragmentation information spanning both the glycan and the peptide moieties. Taken together, these findings illustrate the influence of charge carrier over the energyresolved vibrational activation/dissociation characteristics of glycopeptides, and serve to suggest potential strategies that exploit the analytically useful features uniquely afforded by specific charge carriers or combinations thereof.

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Glycopeptide Analysis, Recent Developments and Applications

Cited by 93 publications

References 48 publications

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

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

Data-independent oxonium ion profiling of multi-glycosylated biotherapeutics

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

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