Microwave-assisted specific chemical digestion for rapid protein identification.
We have developed a rapid microwave-assisted protein digestion technique based on classic acid hydrolysis reaction with 2% formic acid solution. In this mild chemical environment, proteins are hydrolyzed to peptides, which can be directly analyzed by MALDI-MS or ESI-MS without prior sample purification. Dilute formic acid cleaves proteins specifically at the C-terminal of aspartyl (Asp) residues within 10 min of exposure to microwave irradiation. By adjusting the irradiation time, we found that the extent of protein fragmentation can be controlled, as shown by the single fragmentation of myoglobin at the C-terminal of any of the Asp residues. The efficacy and simplicity of this technique for protein identification are demonstrated by the peptide mass maps of in-gel digested myoglobin and BSA, as well as proteins isolated from Escherichia coli K12 cells.
Hua
Lin
L
Genome Institute of Singapore, Singapore.
Low
Teck Yew
TY
Sze
Siu Kwan
SK
D000818:Animals; D002417:Cattle; D004063:Digestion; D048168:Escherichia coli K12 / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005561:Formates / Q000378:metabolism; D006736:Horses; D006868:Hydrolysis; D008872:Microwaves; D009211:Myoglobin / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D029968:Escherichia coli Proteins; D005561:Formates; D009211:Myoglobin; D010446:Peptide Fragments; C030544:formic acid; D012710:Serum Albumin, Bovine