Rapid and efficient protein enzymatic digestion: An experimental comparison

Abstract

The major objective of proteomics is to identify and examine the large numbers of proteins extracted from complex biological systems. This is generally achieved by combining various techniques of protein separation with a mass spectrometric analysis of proteins that are digested enzymatically. Recently, several alternatives to this standard protocol have been developed for efficient and fast protein digestion. One option is the use of modified trypsin instead of native trypsin for the in-gel digestion of proteins. Microwave, ultrasonic-assisted protein enzymatic digestion and proteolysis accelerated by infrared radiation are other suitable alternatives. The application of the variable performance of the fast enzymatic digestion of proteins by using different techniques is reported here. The advantage of these methods is to have the ability to detect proteins in a shorter span of time. For example, using alternative protein digestion takes only minutes, in contrast to the several hours required by conventional methods. To demonstrate the suitability of this fast procedure, the digestion of carbonic anhydrase, bovine serum albumin, lysozyme and proteins extracted from plants (Hordeum vulgare, Arabidopsis thaliana) were used. Considering that the required reaction time for the conventional method is much longer, these applied methodic approaches tend to give in-gel digestion a much higher efficiency rating. This study examines the fast, efficient and low-cost proteolytic strategies for the digestion process, and for protein identification based on the use of ultrasound and infrared technology. In addition, comparisons of the applied techniques were studied. Several differences were found, suggesting the potential use of proteolysis accelerated by infrared radiation.

Integration of online digestion and electrolytic reduction with mass spectrometry for rapid disulfide-containing protein structural analysis

“…However, the conventional enzymatic digestion method is slow and requires overnight incubation, which can be another time-consuming step. Protocols for fast protein digestion have been introduced, such as digestion using modified enzymes [31], microwave digestion [32–34], ultrasonic-assisted protein digestion [35–38] and proteolysis accelerated by infrared radiation [38–40]. Results have been shown that protein digestion can be finished in minutes without reducing efficiency and accuracy.…”

Application of the ETD/PTR reactions in top‐down proteomics as a faster alternative to bottom‐up nanoLC‐MS/MS protein identification

“…A protocol has recently been published describing very fast (~10 min) and selective cleavage of proteins by microwave irradiation, but hydrolysis is limited to the N‐terminal peptide bond of aspartic acid residue only. Other methods are also available for the fast cleavage of proteins supported by sonification or infrared irradiation . With the judgment from our experiments, the time necessary for successful identification of longer peptides or small proteins by ETD/PTR reaction is fairly shorter than this on the basis of the protein cleavage with subsequent LC‐MS/MS technique application. Total cost of the equipment.…”

“…Other methods are also available for the fast cleavage of proteins supported by sonification or infrared irradiation. [15] With the judgment from our experiments, the time necessary for successful identification of longer peptides or small proteins by ETD/PTR reaction is fairly shorter than this on the basis of the protein cleavage with subsequent LC-MS/MS technique application. 2.…”

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

“…However, high concentration of trypsin could lead to autodigestion that interferes with target protein analysis (41). To achieve a high digestion efficiency within a short digestion time, several alternative approaches have been developed, including ultrasound-, high pressure-, infrared- and microwave-assisted digestions (42-46), as well as on-column digestion using immobilized enzyme (47, 48). The latter strategy is particularly interesting because it can be performed online with MS.…”