Microwave-assisted nonspecific proteolytic digestion and controlled methylation for glycomics applications

Liu, Xin; Chan, Kalok; Chu, Ivan K.; Jian-jun, LI

doi:10.1016/j.carres.2008.07.010

Cited by 18 publications

(13 citation statements)

References 30 publications

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“…This decrease might be attributed to nonspecific cleavages at high temperatures. This observation is in agreement with the previously published data obtained from microwave‐assisted pronase E digestion of glycoproteins 22…”

Section: Resultssupporting

confidence: 93%

“…Many reactions including tryptic14–17 and acid‐mediated18, 19 proteolysis of proteins, identification of metal‐catalyzed oxidation sites on proteins20 and N ‐linked deglycosylation21 of glycoproteins can be accelerated, made selective, or increased in efficiency when microwave radiation is used as a heat source. A nonspecific glycoproteolytic digestion using pronase E, which usually requires 2–3 days to complete, has been accelerated and the reaction time reduced to minutes using a microwave‐assisted method 22. However, there have not been any accounts to demonstrate that microwave‐assisted tryptic digestion of glycoproteins could be used to accelerate the digestion process of these proteins which might be sterically hindered by the presence of the glycan moieties.…”

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confidence: 99%

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Rapid and efficient glycoprotein identification through microwave‐assisted enzymatic digestion

Segu

Hammad

Mechref

2010

Rapid Comm Mass Spectrometry

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Identification of protein glycosylation sites is analytically challenging due to the diverse glycan structures associated with a glycoprotein. Mass spectrometry (MS)-based identification and characterization of glycoproteins has been achieved predominantly with the bottom-up approach, which typically involves the enzymatic cleavage of proteins to peptides prior to LC/MS or LC/MS/MS analysis. However, the process can be challenging due to the structural variations and steric hindrance imposed by the attached glycans. Alternatives to conventional heating protocols, that increase the rate of enzymatic cleavage of glycoproteins, may aid in addressing these challenges. An enzymatic digestion of a glycoprotein can be accelerated and made more efficient through microwave-assisted digestion. In this paper, a systematic study was conducted to explore the efficiency of microwave-assisted enzymatic (trypsin) digestion (MAED) of glycoproteins as compared with the conventional method. In addition, the optimum experimental parameters for the digestion such as temperature, reaction time, and microwave radiation power were investigated. It was determined that efficient tryptic digestion of glycoproteins was attained in 15 min, allowing comparable if not better sequence coverage through LC/MS/MS analysis. Optimum tryptic cleavage was achieved at 45°C irrespective of the size and complexity of the glycoprotein. Moreover, MAED allowed the detection and identification of more peptides and subsequently higher sequence coverage for all model glycoprotein. MAED also did not appear to prompt a loss or partial cleavage of the glycan moieties attached to the peptide backbones.

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Section: Resultssupporting

confidence: 93%

mentioning

confidence: 99%

Rapid and efficient glycoprotein identification through microwave‐assisted enzymatic digestion

Segu

Hammad

Mechref

2010

Rapid Comm Mass Spectrometry

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“…One of the problems with the pronase approach is the long reaction times (1-3 days) that are required. Liu et al (2008b) have reduced this to as little as 5 min using microwave irradiation. Because of poor signals associated with the presence of basic amino acids, the investigators methylated them with methyl iodide to give a quaternized amine group, thus considerably enhancing the signal.…”

Section: Analysis Of Carbohydrates and Glycoconjugatesmentioning

confidence: 99%

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

Harvey

2011

Mass Spectrometry Reviews

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This review is the fifth update of the original review, published in 1999, on the application of MALDI mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2008. The first section of the review covers fundamental studies, fragmentation of carbohydrate ions, use of derivatives and new software developments for analysis of carbohydrate spectra. Among newer areas of method development are glycan arrays, MALDI imaging and the use of ion mobility spectrometry. The second section of the review discusses applications of MALDI MS to the analysis of different types of carbohydrate. Specific compound classes that are covered include carbohydrate polymers from plants, N- and O-linked glycans from glycoproteins, biopharmaceuticals, glycated proteins, glycolipids, glycosides and various other natural products. There is a short section on the use of MALDI mass spectrometry for the study of enzymes involved in glycan processing and a section on the use of MALDI MS to monitor products of the chemical synthesis of carbohydrates with emphasis on carbohydrate-protein complexes and glycodendrimers. Corresponding analyses by electrospray ionization now appear to outnumber those performed by MALDI and the amount of literature makes a comprehensive review on this technique impractical. However, most of the work relating to sample preparation and glycan synthesis is equally relevant to electrospray and, consequently, those proposing analyses by electrospray should also find material in this review of interest.

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“…For example, Fenselau and coworkers demonstrated microwave-accelerated acidmediated digestion of glycoproteins to generate glycopeptides in minutes [50]. Microwave irradiation has also been shown to accelerate protease digestions, for example, with pronase [51].…”

Section: Other Proteolysis Strategiesmentioning

confidence: 97%

Engaging Challenges in Glycoproteomics: Recent Advances in MS-Based Glycopeptide Analysis

2015

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The proteomic analysis of glycosylation is uniquely challenging. The numerous and varied biological roles of protein-linked glycans have fueled a tremendous demand for technologies that enable rapid, in-depth structural examination of glycosylated proteins in complex biological systems. In turn, this demand has driven many innovations in wide ranging fields of bioanalytical science. This review will summarize key developments in glycoprotein separation and enrichment, glycoprotein proteolysis strategies, glycopeptide separation and enrichment, the role of mass measurement accuracy in glycopeptide detection, glycopeptide ion dissociation methods for MS/MS, and informatic tools for glycoproteomic analysis. In aggregate, this selection of topics serves to encapsulate the present status of MS-based analytical technologies for engaging the challenges of glycoproteomic analysis.

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Microwave-assisted nonspecific proteolytic digestion and controlled methylation for glycomics applications

Cited by 18 publications

References 30 publications

Rapid and efficient glycoprotein identification through microwave‐assisted enzymatic digestion

Rapid and efficient glycoprotein identification through microwave‐assisted enzymatic digestion

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

Engaging Challenges in Glycoproteomics: Recent Advances in MS-Based Glycopeptide Analysis

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