2010
DOI: 10.1016/j.jasms.2009.11.006
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Digestion completeness of microwave-assisted and conventional trypsin-catalyzed reactions

Abstract: Microwave-assisted proteolytic digestion often yields misscleaved peptides, attributed to incomplete hydrolysis reactions between enzymes and substrates. The number of missed cleavages is an important parameter in proteome database searching. This study investigates how various factors affect digestion processes. Optimum conditions for microwave-assisted digestion (50 mM Tris buffer, 30 min at 60°C, and enzyme to protein molar ratio of 1:5) were determined. The digestion products obtained from eight standard p… Show more

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Cited by 46 publications
(31 citation statements)
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“…If the latter is the case, it is important that the protein of interest is completely digested to its peptide fragments. While maximum digestion efficiency can be reached by changing the digestion time or protease concentration, applying microwave-assisted digestion, and adding a different surfactant and solubilizing agents or protease type (Lyc-C plus trypsin), 21,68,72 it is usually difficult to prove whether digestion is complete (100% efficiency). Peptide stability is an important factor determining apparent digestion efficiency, as some peptides degrade faster than others.…”
Section: Incomplete Vs Inconsistent Protein Digestion and The Concepmentioning
confidence: 99%
“…If the latter is the case, it is important that the protein of interest is completely digested to its peptide fragments. While maximum digestion efficiency can be reached by changing the digestion time or protease concentration, applying microwave-assisted digestion, and adding a different surfactant and solubilizing agents or protease type (Lyc-C plus trypsin), 21,68,72 it is usually difficult to prove whether digestion is complete (100% efficiency). Peptide stability is an important factor determining apparent digestion efficiency, as some peptides degrade faster than others.…”
Section: Incomplete Vs Inconsistent Protein Digestion and The Concepmentioning
confidence: 99%
“…Myoglobin, known as a protease-resistant-protein, is more difficult to digest (49, 50). An on-column tryptic digestion was also performed with the use of the same capillary column mentioned above.…”
Section: Resultsmentioning
confidence: 99%
“…Digestion efficiencies were referred to as the ratio of the abundance of the most abundant peptide product to that of this peptide plus the undigested protein. Optimal conditions were found to be microwave-assisted irradiation at 60 °C for 30 min in a 50 mM Tris buffer with a of 1:5 or 1:25 (Reddy et al 2010). It should be noted that this method is incompatible with subsequent MS analysis when Tris is used as a buffer, without a buffer exchange.…”
Section: Microwave Assisted Digestionmentioning
confidence: 99%