Fast Peptide Sequencing without MS/MS? Peptide Primary Structure Elucidation by Mass Spectrometry using Partial Hydrolysis

Abstract

A straightforward procedure for the primary structure elucidation of peptides is presented. It is found that hydrolysis attacks only the N- and C-ends of peptides leading to partial cleavage of adjacent amino acids. Depending on the peptide, either a single or two overlapping peptide ladders are created which can be analysed using a simple mass spectrometer such as a linear time-of-flight mass spectrometer (TOF-MS) with no MS/MS capabilities.The procedure is demonstrated on the peptide Melittin, a 26 amino acid single chain peptide. Melittin was partially hydrolysed. After only 1 min of hydrolysis time two peptide ladders were already fully developed and could be analysed. It was found that Melittin is cleaved from both the C- and the N-terminus. About 80% of the primary structure can be determined from the mass spectrum obtained by MALDI-linear TOF-MS. Advantages of the methodology described are its simplicity and quickness and the fact that there is no need for high-performance mass spectrometers.Besides its analytical value, the partial hydrolysis procedure can be used for educational purposes such as lab courses for students at Universities of Applied Sciences to introduce concepts of modern peptide structure analysis in combination with mass spectrometry.