Characterizing <i>O</i>-Linked Glycopeptides by Electron Transfer Dissociation: Fragmentation Rules and Applications in Data Analysis

Zhu, Zhongkui; Su, Xiaomeng; Clark, Daniel F.; Go, Eden P.; Desaire, Heather

doi:10.1021/ac401814h

Cited by 47 publications

(39 citation statements)

References 41 publications

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“…The following gradient was used: 5% eluent B for 5 min, followed by a linear increase to 40% B in 50 min, and a ramp to 95% B in 10 min. The column was held at 95% B for another 10 min before re-equilibration [14-15]. The mass spectrometer was operated at an ESI spray voltage of 3.0 kV with the capillary temperature of 250 °C, and full scan mass spectra ( m/z 400-2000) were collected at a resolution of 30,000 at m/z 400.…”

Section: Methodsmentioning

confidence: 99%

Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS

Zhu

Desaire

2014

J. Am. Soc. Mass Spectrom.

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N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs. To overcome this limitation, we developed a new method to measure the glycosylation site occupancy that does not rely on converting glycosylated peptides to their deglycosylated forms. Specifically, the overall protein concentration and the non-glycosylated portion of the protein are quantified simultaneously by using heavy isotope-labeled internal standards coupled with LC-MS analysis, and the extent of site occupancy is accurately determined. The efficacy of the method was demonstrated by quantifying the occupancy of a glycosylation site on bovine fetuin. The developed method is the first work that measures the glycosylation site occupancy without using PNGase F, and it can be done in parallel with glycopeptide analysis because the glycan remains intact throughout the workflow.

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Section: Methodsmentioning

confidence: 99%

Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS

Zhu

Desaire

2014

J. Am. Soc. Mass Spectrom.

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“…In view of the great complementarity possible with ETD, a few programs have included functionality to analyze the ETD data of glycopeptides (97,(115)(116)(117)(118). This functionality is particularly useful in O-linked glycopeptide analysis, because no consensus sequence exists to predict the potential O-linked glycosylation site, which is also hard to determine by CID data (119-121).…”

Section: Automated Glycoproteomicsmentioning

confidence: 99%

“…The software allows merging of CID and ETD search results, and users can compare different modification site assignments for site localization. To automate the glycopeptide identification workflow by ETD, Zhu et al (97,117) studied the fragmentation patterns of N-and O-linked glycopeptides from model glycoproteins and developed novel algorithms to consider multiple ion series (c, z, and y ions) of putative glycopeptide candidates separately. By implementing the algorithms into standalone software programs that handle glycopeptide ETD data, the correct glycopeptide compositions and site assignments were made with high throughput and high accuracy.…”

Section: Automated Glycoproteomicsmentioning

confidence: 99%

Carbohydrates on Proteins: Site-Specific Glycosylation Analysis by Mass Spectrometry

Zhu

Desaire²

2015

Annual Rev. Anal. Chem.

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Glycosylation on proteins adds complexity and versatility to these biologically vital macromolecules. To unveil the structure-function relationship of glycoproteins, glycopeptide-centric analysis using mass spectrometry (MS) has become a method of choice because the glycan is preserved on the glycosylation site and site-specific glycosylation profiles of proteins can be readily determined. However, glycopeptide analysis is still challenging given that glycopeptides are usually low in abundance and relatively difficult to detect and the resulting data require expertise to analyze. Viewing the urgent need to address these challenges, emerging methods and techniques are being developed with the goal of analyzing glycopeptides in a sensitive, comprehensive, and high-throughput manner. In this review, we discuss recent advances in glycoprotein and glycopeptide analysis, with topics covering sample preparation, analytical separation, MS and tandem MS techniques, as well as data interpretation and automation.

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“…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). 32,33 One useful outcome of these investigations has been the observation of a high degree of complementarity between the vibrational activation/dissociation spectra and the electron capture/transfer dissociation spectra of glycopeptide ions.…”

Section: Introductionmentioning

confidence: 99%

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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Characterizing O-Linked Glycopeptides by Electron Transfer Dissociation: Fragmentation Rules and Applications in Data Analysis

Cited by 47 publications

References 41 publications

Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS

Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS

Carbohydrates on Proteins: Site-Specific Glycosylation Analysis by Mass Spectrometry

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

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