Systematic characterization of extracellular glycoproteins using mass spectrometry

Sun, Fangxu; Suttapitugsakul, Suttipong; Wu, Ronghu

doi:10.1002/mas.21708

Cited by 12 publications

(7 citation statements)

References 220 publications

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“…Protein glycosylation is often analyzed by highperformance liquid chromatography (HPLC)-MS/MS [49][50][51]. To identify hydrophilic glycopeptides, it is necessary to enrich them from peptides or other nonglycopeptide components [52].…”

Section: Different Types Of Glycopeptide Enrichment Methodsmentioning

confidence: 99%

Advances in glycopeptide enrichment methods for the analysis of protein glycosylation over the past decade

Zhang

et al. 2022

J of Separation Science

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Advances in bioanalytical technology have accelerated the analysis of complex protein glycosylation, which is beneficial to understand glycosylation in drug discovery and disease diagnosis. Due to its biological uniqueness in the course of disease occurrence and development, disease‐specific glycosylation requires quantitative characterization of protein glycosylation. We provide a comprehensive review of recent advances in glycosylation analysis, including workflows for glycoprotein digestion, glycopeptide separation and enrichment, and mass spectrometry sequencing. We specifically focus on different strategies for glycopeptide enrichment through physical interaction, chemical oxidation, or metabolic labeling of intact glycopeptides. Recent advances and challenges of O‐glycosylation analysis are presented, and the development of improved enrichment methods combining different proteases to analyze O‐glycosylation is also proposed.

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Section: Different Types Of Glycopeptide Enrichment Methodsmentioning

confidence: 99%

Advances in glycopeptide enrichment methods for the analysis of protein glycosylation over the past decade

Zhang

et al. 2022

J of Separation Science

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“…Compared to the whole cell glycoproteins, cell membrane glycoproteins are less abundant, and are easily confused with intracellular proteins. The enrichment of surface glycoproteins is of great significance for comprehensive analysis by MS. To better investigate the glycosylation on the cell membrane, researchers use density-gradient centrifugation to separate the plasma membrane from the cell, especially sucrose-gradient centrifugation is the most widely used method, which is also a classic method for membrane protein separation ( 92 , 93 ). However, ultracentrifugation does not separate membrane glycoproteins but all membrane proteins, and this method cannot completely eliminate cytoplasmic proteins ( 93 ).…”

Section: Recent Advances In Glycomics-based Biomarker Discoverymentioning

confidence: 99%

“…The enrichment of surface glycoproteins is of great significance for comprehensive analysis by MS. To better investigate the glycosylation on the cell membrane, researchers use density-gradient centrifugation to separate the plasma membrane from the cell, especially sucrose-gradient centrifugation is the most widely used method, which is also a classic method for membrane protein separation ( 92 , 93 ). However, ultracentrifugation does not separate membrane glycoproteins but all membrane proteins, and this method cannot completely eliminate cytoplasmic proteins ( 93 ). In recent years, with the development of chemical biology and MS-based proteomics, more precise analysis of cell surface glycoproteins has become possible.…”

Section: Recent Advances In Glycomics-based Biomarker Discoverymentioning

confidence: 99%

“…Currently, few unnatural carbohydrate metabolism precursors are widely used, which greatly limits the systematic research of glycosylation in a cell membrane protein ( 93 ). Unnatural sugar incorporation relies on competing cellular metabolic processes with natural sugars, which is inefficient and time-consuming.…”

Section: Recent Advances In Glycomics-based Biomarker Discoverymentioning

confidence: 99%

“…Unnatural sugar incorporation relies on competing cellular metabolic processes with natural sugars, which is inefficient and time-consuming. The use of glycosyltransferases and nucleotide-sugar analogs to directly label cell surface glycans was also pursued by researchers ( 93 ). Selective exo-enzymatic labeling (SEEL) method was developed to efficiently label cell surface glycans with recombinant sialyltransferases and nucleotide-Sia analogs.…”

Section: Recent Advances In Glycomics-based Biomarker Discoverymentioning

confidence: 99%

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Cancer glycomics offers potential biomarkers and therapeutic targets in the framework of 3P medicine

et al. 2022

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Glycosylation is one of the most important post-translational modifications (PTMs) in a protein, and is the most abundant and diverse biopolymer in nature. Glycans are involved in multiple biological processes of cancer initiation and progression, including cell-cell interactions, cell-extracellular matrix interactions, tumor invasion and metastasis, tumor angiogenesis, and immune regulation. As an important biomarker, tumor-associated glycosylation changes have been extensively studied. This article reviews recent advances in glycosylation-based biomarker research, which is useful for cancer diagnosis and prognostic assessment. Truncated O-glycans, sialylation, fucosylation, and complex branched structures have been found to be the most common structural patterns in malignant tumors. In recent years, immunochemical methods, lectin recognition-based methods, mass spectrometry (MS)-related methods, and fluorescence imaging-based in situ methods have greatly promoted the discovery and application potentials of glycomic and glycoprotein biomarkers in various cancers. In particular, MS-based proteomics has significantly facilitated the comprehensive research of extracellular glycoproteins, increasing our understanding of their critical roles in regulating cellular activities. Predictive, preventive and personalized medicine (PPPM; 3P medicine) is an effective approach of early prediction, prevention and personalized treatment for different patients, and it is known as the new direction of medical development in the 21st century and represents the ultimate goal and highest stage of medical development. Glycosylation has been revealed to have new diagnostic, prognostic, and even therapeutic potentials. The purpose of glycosylation analysis and utilization of biology is to make a fundamental change in health care and medical practice, so as to lead medical research and practice into a new era of 3P medicine.

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A Systematic Investigation of Proteoforms with N‐Terminal Glycine and Their Dynamics Reveals Its Impacts on Protein Stability

Xu,

Wang

et al. 2024

Angewandte Chemie

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The N‐termini of proteins can regulate their degradation, and the same protein with different N‐termini may have distinct dynamics. Recently, it was found that N‐terminal glycine can serve as a degron recognized by two E3 ligases, but N‐terminal glycine was also reported to stabilize proteins. Here we developed a chemoenzymatic method for selective enrichment of proteoforms with N‐terminal glycine and integrated dual protease cleavage to further improve the enrichment specificity. Over 2000 unique peptides with protein N‐terminal glycine were analyzed from >1000 proteins in human cells, and most of them are previously unknown, indicating the effectiveness of the current method to capture low‐abundance proteoforms with N‐terminal glycine. The degradation rates of proteoforms with N‐terminal glycine were quantified along with those of proteins from the whole proteome. Bioinformatic analyses reveal that proteoforms with N‐terminal glycine with the fastest and slowest degradation rates have different functions and localizations. Membrane proteins with N‐terminal glycine and proteins with N‐terminal glycine from the N‐terminal methionine excision degrade more rapidly. Furthermore, the secondary structures, adjacent amino acid residues, and protease specificities for N‐terminal glycine are also vital for protein degradation. The results advance our understanding of the effects of N‐terminal glycine on protein properties and functions.

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Systematic characterization of extracellular glycoproteins using mass spectrometry

Cited by 12 publications

References 220 publications

Advances in glycopeptide enrichment methods for the analysis of protein glycosylation over the past decade

Advances in glycopeptide enrichment methods for the analysis of protein glycosylation over the past decade

Cancer glycomics offers potential biomarkers and therapeutic targets in the framework of 3P medicine

A Systematic Investigation of Proteoforms with N‐Terminal Glycine and Their Dynamics Reveals Its Impacts on Protein Stability

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