Rapid online nonenzymatic protein digestion combining microwave heating acid hydrolysis and electrochemical oxidation.
We report an online nonenzymatic method for site-specific digestion of proteins to yield peptides that are well suited for collision-induced dissociation tandem mass spectrometry. The method combines online microwave heating acid hydrolysis at aspartic acid and online electrochemical oxidation at tryptophan and tyrosine. The combined microwave/electrochemical digestion is reproducible and produces peptides with an average sequence length of 10 amino acids. This peptide length is similar to the average peptide length of 9 amino acids obtained by digestion of proteins with the enzyme trypsin. As a result, the peptides produced by this novel nonenzymatic digestion method, when analyzed by electrospray ionization mass spectrometry, produce protonated molecules with mostly +1 and +2 charge states. The combination of these two nonenzymatic methods overcomes shortcomings with each individual method in that (i) peptides generated by the microwave-hydrolysis method have an average amino acid length of 16 amino acids and (ii) the electrochemical-cleavage method is unable to reproducibly digest proteins with molecular masses above 4 kDa. Preliminary results are presented on the application and utility of this rapid online digestion (total of 6 min of digestion time) on a series of standard peptides and proteins as well as an Escherichia coli protein extract.
Department of Chemistry, 1000 East University Avenue (Department 3838), University of Wyoming, Laramie, Wyoming 82071, United States. basile@uwyo.edu
Franco
F
Basile
Nicolas
N
Hauser
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D004220:Disulfides / Q000737:chemistry; D004563:Electrochemistry; D004926:Escherichia coli / Q000166:cytology; D006358:Hot Temperature; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008872:Microwaves; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D004220:Disulfides; D010446:Peptide Fragments; D011506:Proteins