Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry.
Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein.
Reiz
Bela
B
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
Li
Liang
L
D000143:Acids / Q000737:chemistry; D000468:Alkalies / Q000737:chemistry; D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry*; D006868:Hydrolysis; D008872:Microwaves; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000143:Acids; D000468:Alkalies; D004220:Disulfides; D011506:Proteins; D009113:Muramidase