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

Zhu, Zhongkui; Go, Eden P.; Desaire, Heather

doi:10.1007/s13361-014-0859-2

Cited by 29 publications

(26 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Collision-induced dissociation (CID), a collisionbased activation method available in most commercial instruments including ion trap (64), triple quadrupole (76), quadrupole TOF (77), and other hybrids (78)(79)(80), is routinely used in MS/MS study of glycopeptides. …”

Section: Collisional Dissociation Methodsmentioning

confidence: 99%

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

Zhu

Desaire²

2015

Annual Rev. Anal. Chem.

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Collisional Dissociation Methodsmentioning

confidence: 99%

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

Zhu

Desaire²

2015

Annual Rev. Anal. Chem.

Self Cite

View full text Add to dashboard Cite

show abstract

“…As discussed by Desaire and coworkers [39], spontaneous deamidation of asparagine side chains occurring in the context of a potential N-glycosylation site can lead to an overestimation of site occupancy when all such deamidation is presumed to be attributable to glycan release Fig. 4 Quantitative analysis of transferrin site specific occupancy in patients with CDG-I diseases.…”

Section: Glycosylation Site-specific Occupancy Approachesmentioning

confidence: 99%

“…Consequently, the two peptides will have different ionization efficiencies, further contributing to errors in the quantitation of site occupancy. As an alternative approach to circumvent such limitations, Zhu et al [39] implemented a scheme based on the use of two isotopically labeled standard peptides (Fig. 5).…”

Section: Glycosylation Site-specific Occupancy Approachesmentioning

confidence: 99%

“…Peptide standards of the same sequence were heavy isotope-labeled and spiked into the mixture to allow for quantification of the total protein concentration and sitespecific non-occupancy. Reproduced with permission from Zhu et al [39], copyright 2014 American Society for Mass Spectrometry new approaches to effective and efficient analysis of intact glycopeptides [50][51][52][53][54][55][56].…”

Section: Glycosite-specific Glycosylation Approachesmentioning

confidence: 99%

See 1 more Smart Citation

A case for protein-level and site-level specificity in glycoproteomic studies of disease

Schumacher

Dodds

2016

Glycoconj J

View full text Add to dashboard Cite

Abnormal glycosylation of proteins is known to be either resultant or causative of a variety of diseases. This makes glycoproteins appealing targets as potential biomarkers and focal points of molecular studies on the development and progression of human ailment. To date, a majority of efforts in disease glycoproteomics have tended to center on either determining the concentration of a given glycoprotein, or on profiling the total population of glycans released from a mixture of glycoproteins. While these approaches have demonstrated some diagnostic potential, they are inherently insensitive to the fine molecular detail which distinguishes unique and possibly disease relevant glycoforms of specific proteins. As a consequence, such analyses can be of limited sensitivity, specificity, and accuracy because they do not comprehensively consider the glycosylation status of any particular glycoprotein, or of any particular glycosylation site. Therefore, significant opportunities exist to improve glycoproteomic inquiry into disease by engaging in these studies at the level of individual glycoproteins and their exact loci of glycosylation. In this concise review, the rationale for glycoprotein and glycosylation site specificity is developed in the context of human disease glycoproteomics with an emphasis on N-glycosylation. Recent examples highlighting disease-related perturbations in glycosylation will be presented, including those involving alterations in the overall glycosylation of a specific protein, alterations in the occupancy of a given glycosylation site, and alterations in the compositional heterogeneity of glycans occurring at a given glycosylation site. Each will be discussed with particular emphasis on how protein-specific and site-specific approaches can contribute to improved discrimination between glycoproteomes and glycoproteins associated with healthy and unhealthy states.

show abstract

“…In addition, antibodies are expensive and the experimental procedures are typically time-consuming and laborintensive. Alternatively, MS-based techniques can assist in the confident global identification and quantification of proteins without the use of antibodies [11][12][13].…”

Section: Introductionmentioning

confidence: 99%

Mass Spectrometric Analysis of the Cell Surface N-Glycoproteome by Combining Metabolic Labeling and Click Chemistry

Smeekens

Chen

2014

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

Abstract. Cell surface N-glycoproteins play extraordinarily important roles in cell-cell communication, cell-matrix interactions, and cellular response to environmental cues. Global analysis is exceptionally challenging because many N-glycoproteins are present at low abundances and effective separation is difficult to achieve. Here, we have developed a novel strategy integrating metabolic labeling, copper-free click chemistry, and mass spectrometry (MS)-based proteomics methods to analyze cell surface N-glycoproteins comprehensively and site-specifically. A sugar analog containing an azido group, N-azidoacetylgalactosamine, was fed to cells to label glycoproteins. Glycoproteins with the functional group on the cell surface were then bound to dibenzocyclooctyne-sulfo-biotin via copper-free click chemistry under physiological conditions. After protein extraction and digestion, glycopeptides with the biotin tag were enriched by NeutrAvidin conjugated beads. Enriched glycopeptides were deglycosylated with peptide-N-glycosidase F in heavy-oxygen water, and in the process of glycan removal, asparagine was converted to aspartic acid and tagged with 18 O for MS analysis. With this strategy, 144 unique N-glycopeptides containing 152 Nglycosylation sites were identified in 110 proteins in HEK293T cells. As expected, 95% of identified glycoproteins were membrane proteins, which were highly enriched. Many sites were located on important receptors, transporters, and cluster of differentiation proteins. The experimental results demonstrated that the current method is very effective for the comprehensive and site-specific identification of the cell surface N-glycoproteome and can be extensively applied to other cell surface protein studies.

show abstract

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

Cited by 29 publications

References 17 publications

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

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

A case for protein-level and site-level specificity in glycoproteomic studies of disease

Mass Spectrometric Analysis of the Cell Surface N-Glycoproteome by Combining Metabolic Labeling and Click Chemistry

Contact Info

Product

Resources

About