Assessment of microwave‐assisted enzymatic digestion by measuring glycated hemoglobin A1c by mass spectrometry

Vesper, Hubert W.; Mi, Luchuan; Archibold, Enada; Myers, Gary L.

doi:10.1002/rcm.2135

Cited by 40 publications

(25 citation statements)

References 19 publications

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“…A quantitative assessment of microwave-assisted enzymatic digestion, using trypsin and endoproteinase Glu-C (Glu-C), was performed on glycated hemoglobin A1c by Vesper et al (2005). An LCQ Deca ion trap mass spectrometer was used for qualitative analysis and a TSQ Quantum triple quadrupole mass spectrometer for quantitation of peptides that corresponded to the tryptic and Glu-C digestion products from the N-terminus.…”

Section: A Proteolytic Enzymesmentioning

confidence: 99%

“…However, for biological enzymes, superheating would not be an apt theory, because these molecules are very sensitive and often heat-labile. Also, proteins are much larger molecules, have Vesper et al (2005) that the heat-labile enzyme Glu-C has decreased proteolysis with microwave-assisted incubation, because the excess energy actually renders this fragile enzyme inert. In the case of MAAH for cleavage of the termini, or acid labile sites within the protein, microwave irradiation may assist in protein solubilization, therefore bringing the protein molecule in close contact with TFA, which may contribute to its rapid hydrolysis.…”

Section: Mechanism Of Catalysismentioning

confidence: 99%

“…However, there are several examples in the literature where a true direct comparison between the microwaveassisted catalysis and the water bath-mediated incubation have been performed at identical temperatures, vessels, and time points Lin et al, 2005;Vesper et al, 2005;Sandoval et al, 2007). Sandoval et al described attempts to prove or disprove the non-thermal effects theory by heating vessels and buffers to the same temperature prior to incubation (microwave vs. water bath).…”

Section: Mechanism Of Catalysismentioning

confidence: 99%

“…Although Pramanik et al state that ''No microwave-assisted reaction is slower than the corresponding conductivity/convection reaction'' , this statement may be the case for the majority of chemical cleavages of proteins and PTMs, but it is not necessarily true for heat-labile enzymes, for example, Glu-C (Vesper et al, 2005). When considering the use of microwaveassistance to increase throughput, each enzymatic or chemical preparation should be fully investigated on a case-by-case basis.…”

Section: Mechanism Of Catalysismentioning

confidence: 99%

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Microwave‐assisted proteomics

Lill¹,

Ingle²,

Liu³

et al. 2007

Mass Spectrometry Reviews

144

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State-of-the-art proteomic analysis has recently undergone a rapid evolution; with more high-throughput analytical instrumentation and informatic tools available, sample preparation is becoming one of the rate-limiting steps in protein characterization workflows. Recently several protocols have appeared in the literature that employ microwave irradiation as a tool for the preparation of biological samples for subsequent mass spectrometric characterization. Techniques for microwave-assisted biocatalyzed reactions (including sample reduction and alkylation, enzymatic and chemical digestion, removal and analysis of posttranslational modifications and characterization of enzymes and protein-interaction sites) are described. This review summarizes the various approaches undertaken, instrumentation employed, and reduction in overall experimental time observed when microwave assistance is applied. #

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Section: A Proteolytic Enzymesmentioning

confidence: 99%

Section: Mechanism Of Catalysismentioning

confidence: 99%

Section: Mechanism Of Catalysismentioning

confidence: 99%

Section: Mechanism Of Catalysismentioning

confidence: 99%

See 2 more Smart Citations

Microwave‐assisted proteomics

Lill¹,

Ingle²,

Liu³

et al. 2007

Mass Spectrometry Reviews

144

View full text Add to dashboard Cite

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“…45 Havlis et al reported that modification of trypsin by reductive methylation reduced the autolysis and shifted its working optimum to a higher temperature. 44 Thus, mass spectrometric analysis revealed that the samples were not contaminated with trypsin autolysis products, despite the high concentration of this enzyme, whereas the substrates were digested effectively (data not shown).…”

Section: Page Analysis Of Protein Digestion At Different Concentratiomentioning

confidence: 99%

Digestion of Native Proteins for Proteomics Using a Thermocycler

et al. 2008

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Efficient protein digestion is a critical step for successful mass spectrometry analysis. Here we describe simultaneous tryptic digestion and gradual unfolding of native proteins by application of a temperature gradient using a single cycle of 5 min or less in a PCR thermocycler. Chemicals typically used for chromatographic techniques did not affect the digestion efficiency. Tryptic digestion was performed in a small volume (3 μL) with 1.5 μg of trypsin without denaturing agents. This rapid procedure yielded more peptides than conventional methods utilizing chemical denaturation for 18 proteins out of 20. Samples were directly spotted on the MALDI-TOF target plate, without additional purification, thus reducing losses on reversed phase resins.Mass spectrometry (MS) has become an indispensable technique in protein research. 1-4 Usually, denatured proteins are digested with a proteinase and the peptides can then be analyzed by MS. The mass spectrum is compared with the peptide mass fingerprints in databases for protein identification. 1-4 Such mass mapping requires purified proteins. Therefore, this technique is usually used in conjunction with protein fractionation methods such as liquid chromatography (LC) or one-and two-dimensional gel electrophoresis (1DE/2DE). Today multidimensional chromatography and analysis techniques based on computer-controlled HPLC systems are being adopted by industry and academia. 1,5-8 MDCA generally processes proteins in their native conformations rather than denatured as in SDS gels. Native proteins are also isolated by bimolecular interaction analysis instruments, such as BIAcore, which utilize the biological specificity of native proteins for other proteins or DNA for isolation of proteins from complex mixtures. 9-12 Subsequent chemical denaturation protocols 12,13 have been applied to the output of these separation techniques followed by conventional overnight trypsin digestion, with subsequent solid-phase capture and elution of samples for MS analysis.The use of direct protease digestion of native proteins from liquid chromatography or BIAcore would be a great advantage for further analysis by MS. Automation of the integrative LC-MS analysis is currently difficult mainly due to the complicated process of protein digestion. Some proteins are susceptible to proteinases and can be digested under standard conditions (37 °C, neutral pH, etc.), whereas most proteins are not digested effectively. To achieve efficient digestion, those proteins require a pretreatment step that denatures the protein prior to proteinase digestion. Proteins are usually unfolded using chemical denaturants. 13-15 Denaturation at 90 °C has been reported as an alternative to chemical denaturation of proteins. 15 Such denatured proteins are generally aggregated, forming large clumps of protein(s). The process of digestion of such aggregates is very slow (typically overnight digestion is required).*To whom correspondence should be addressed. E-mail: ot16@leicester.ac.uk. It is in the bounds of possibilit...

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