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

Abstract

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 3M 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 0.5μg (7.5pmol) 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. Protein terminal truncation or modification plays an important role in determining the biological functions of a protein. Detailed characterization of protein terminal sequences is critical in biological studies as well as in the development and quality control of protein-based therapeutics and vaccines. In this work, we report a relatively simple method for analyzing protein terminal sequences based on microwave-assisted acid hydrolysis to generate the peptide ladder of a protein, liquid chromatography fractionation of the resultant ladder to collect the low-mass-peptide fraction which mainly contains terminal peptides, and LC-ESI MS/MS sequencing of the collected peptides. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes?