Microwave-assisted acid hydrolysis of proteins combined with peptide fractionation and mass spectrometry analysis for characterizing protein terminal sequences

摘要

We report a relatively simple mass spectrometric technique for characterizing the terminal amino acid sequences of proteins. It is based on the use of microwave-assisted acid hydrolysis (MAAH) with 3 M HCl to hydrolyze a protein into polypeptide ladders with varying sizes of up to the molecular mass of the protein. The hydrolysate is, then fractionated by isocratic reversed phase liquid chromatography (RPLC) to produce a low-mass-peptide fraction mainly consisting of the terminal peptides. This fraction is subjected to LC tandem mass spectrometry (MS/MS) analysis to generate the terminal peptide sequence information. Using bovine serum albumin as an example, it is shown that more than 10 terminal peptides of each end could be identified using as little as 0.5 mug (7.5 pmol) of protein. This method was applied for the characterization of a recombinant protein (mCherry with an additional sequence tag added to the N-terminal for expression and purification) and its truncated form (mCherry treated with enterokinase to cleave off the tag). Sequence errors and unexpected by-products with different terminal sequences were determined from these two samples, illustrating that this method of HCl MAAH with peptide fractionation and LC-MS/MS analysis should be useful for detailed characterization of protein terminal sequences.