title: 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.
authors.affiliation: Institute of Analytical Chemistry of the ASCR, Brno, Czech Republic.
authors.forename: Filip
authors.initials: F
authors.lastname: Dycka
authors.forename: Pavel
authors.initials: P
authors.lastname: Bobal
authors.forename: Karel
authors.initials: K
authors.lastname: Mazanec
authors.forename: Janette
authors.initials: J
authors.lastname: Bobalova
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D010940:Plant Proteins; D011506:Proteins; D014357:Trypsin