MS-based proteomics for comprehensive investigation of protein<i>O</i>-GlcNAcylation

Xu, Senhan; Sun, Fangxu; Tong, Ming Him; Wu, Ronghu

doi:10.1039/d1mo00025j

Cited by 7 publications

(4 citation statements)

References 105 publications

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“…To enrich O -GlcNAcylated proteins, a sugar analogue containing an azide group was used to label glycoproteins. The sugar analogue is structurally similar to its natural counterpart and can be incorporated into glycoproteins. − In this work, Ac 4 GalNAz was used as it has been widely applied to label O -GlcNAcylated proteins. − The azide group enables further enrichment of labeled glycoproteins using click chemistry. Moreover, newly synthesized glycoproteins labeled with Ac 4 GalNAz carrying an azide group are different from existing glycoproteins, ensuring only newly synthesized O -GlcNAcylated proteins/peptides being enriched and analyzed.…”

Section: Resultsmentioning

confidence: 99%

Combining Selective Enrichment and a Boosting Approach to Globally and Site-Specifically Characterize Protein Co-translational O-GlcNAcylation

Yin

2023

Anal. Chem.

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Protein O-GlcNAcylation plays extremely important roles in mammalian cells, regulating signal transduction and gene expression. This modification can happen during protein translation, and systematic and site-specific analysis of protein co-translational O-GlcNAcylation can advance our understanding of this important modification. However, it is extraordinarily challenging because normally O-GlcNAcylated proteins are very low abundant and the abundances of co-translational ones are even much lower. Here, we developed a method integrating selective enrichment, a boosting approach, and multiplexed proteomics to globally and site-specifically characterize protein co-translational O-GlcNAcylation. The boosting approach using the TMT labeling dramatically enhances the detection of co-translational glycopeptides with low abundance when enriched O-GlcNAcylated peptides from cells with a much longer labeling time was used as a boosting sample. More than 180 co-translational O-GlcNAcylated proteins were site-specifically identified. Further analyses revealed that among co-translational glycoproteins, those related to DNA binding and transcription are highly overrepresented using the total identified O-GlcNAcylated proteins in the same cells as the background. Compared with the glycosylation sites on all glycoproteins, co-translational sites have different local structures and adjacent amino acid residues. Overall, an integrative method was developed to identify protein co-translational O-GlcNAcylation, which is very useful to advance our understanding of this important modification.

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Section: Resultsmentioning

confidence: 99%

Combining Selective Enrichment and a Boosting Approach to Globally and Site-Specifically Characterize Protein Co-translational O-GlcNAcylation

Yin

2023

Anal. Chem.

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“…O-GlcNAcylation, the addition of GlcNAc to Ser or Thr, can be used as an indicator of cellular metabolism and has been studied by MS in earlier studies on diabetes (Majumdar et al, 2006;Ma and Hart, 2013;Lim et al, 2014). The considerations needed in studying the small O-GlcNAc modification, including enzymatic analysis methods, have been thoroughly reviewed recently (Maynard and Chalkley, 2021;Xu et al, 2021).…”

Section: Trends In the Reviewed Studies Workflow And Analyte Considerationsmentioning

confidence: 99%

Recent Advances in Mass Spectrometry-Based Glycomic and Glycoproteomic Studies of Pancreatic Diseases

Tabang

Ford

2021

Front. Chem.

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Modification of proteins by glycans plays a crucial role in mediating biological functions in both healthy and diseased states. Mass spectrometry (MS) has emerged as the most powerful tool for glycomic and glycoproteomic analyses advancing knowledge of many diseases. Such diseases include those of the pancreas which affect millions of people each year. In this review, recent advances in pancreatic disease research facilitated by MS-based glycomic and glycoproteomic studies will be examined with a focus on diabetes and pancreatic cancer. The last decade, and especially the last five years, has witnessed developments in both discovering new glycan or glycoprotein biomarkers and analyzing the links between glycans and disease pathology through MS-based studies. The strength of MS lies in the specificity and sensitivity of liquid chromatography-electrospray ionization MS for measuring a wide range of biomolecules from limited sample amounts from many sample types, greatly enhancing and accelerating the biomarker discovery process. Furthermore, imaging MS of glycans enabled by matrix-assisted laser desorption/ionization has proven useful in complementing histology and immunohistochemistry to monitor pancreatic disease progression. Advances in biological understanding and analytical techniques, as well as challenges and future directions for the field, will be discussed.

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“…With the development of MS‐based proteomics, site‐specific analysis of surface glycoproteins has been growing rapidly. It not only provides valuable information about the glycosylation sites but also offers direct experimental evidence for the identification of glycopeptides and glycoproteins (Sun & Wu, 2019; Xiao & Wu, 2017c; Xiao et al, 2018c; Xu et al, 2021). Our group has developed an effective method to comprehensively and site‐specifically investigate glycoproteins located on the cell surface by combining metabolic labeling, bioorthogonal chemistry, and MS‐based proteomics (Figure 3) (Chen et al, 2015; Smeekens et al, 2015; Xiao et al, 2016b).…”

Section: Comprehensive Analysis Of Glycoproteins On the Cell Surface ...mentioning

confidence: 99%

Systematic characterization of extracellular glycoproteins using mass spectrometry

Sun

Suttapitugsakul

2021

Mass Spectrometry Reviews

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Surface and secreted glycoproteins are essential to cells and regulate many extracellular events. Because of the diversity of glycans, the low abundance of many glycoproteins, and the complexity of biological samples, a system‐wide investigation of extracellular glycoproteins is a daunting task. With the development of modern mass spectrometry (MS)‐based proteomics, comprehensive analysis of different protein modifications including glycosylation has advanced dramatically. This review focuses on the investigation of extracellular glycoproteins using MS‐based proteomics. We first discuss the methods for selectively enriching surface glycoproteins and investigating protein interactions on the cell surface, followed by the application of MS‐based proteomics for surface glycoprotein dynamics analysis and biomarker discovery. We then summarize the methods to comprehensively study secreted glycoproteins by integrating various enrichment approaches with MS‐based proteomics and their applications for global analysis of secreted glycoproteins in different biological samples. Collectively, MS significantly expands our knowledge of extracellular glycoproteins and enables us to identify extracellular glycoproteins as potential biomarkers for disease detection and drug targets for disease treatment.

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MS-based proteomics for comprehensive investigation of proteinO-GlcNAcylation

Abstract: Protein O-GlcNAcylation refers to the covalent binding of a single N-acetylglucosamine (GlcNAc) to the serine or threonine residue. This modification primarily occurs on proteins in the nucleus and the cytosol,...

Cited by 7 publications

References 105 publications

Combining Selective Enrichment and a Boosting Approach to Globally and Site-Specifically Characterize Protein Co-translational O-GlcNAcylation

Combining Selective Enrichment and a Boosting Approach to Globally and Site-Specifically Characterize Protein Co-translational O-GlcNAcylation

Recent Advances in Mass Spectrometry-Based Glycomic and Glycoproteomic Studies of Pancreatic Diseases

Systematic characterization of extracellular glycoproteins using mass spectrometry

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