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 proteins were characterized based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Experimental results indicate that the digestion temperature, reaction time, enzyme to substrate ratio, and digestion buffer affect the number of misscleaved peptides and incomplete digestion percentages. Although all protein molecules in a sample could be digested into peptides within a few minutes under microwave irradiation, longer reaction times or methods to maximize the enzyme activity should be considered if digestion completeness is a major concern. (J Am Soc Mass Spectrom 2010, 21, 421-424)

Critical Issues and Optimized Practices in Quantification of Protein Abundance Level to Determine Interindividual Variability in DMET Proteins by LC‐MS/MS Proteomics

“…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.…”

Enhancing performance of liquid sample desorption electrospray ionization mass spectrometry using trap and capillary columns

“…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.…”

A Critical Review of Trypsin Digestion for LC-MS Based Proteomics

“…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.…”