Subcritical Water Hydrolysis of Peptides: Amino Acid Side-Chain Modifications

Thomas Powell,Steve Bowra,Helen J Cooper

doi:10.1007/s13361-017-1676-1

Abstract

Previously we have shown that subcritical water may be used as an alternative to enzymatic digestion in the proteolysis of proteins for bottom-up proteomics. Subcritical water hydrolysis of proteins was shown to result in protein sequence coverages greater than or equal to that obtained following digestion with trypsin; however, the percentage of peptide spectral matches for the samples treated with trypsin were consistently greater than for those treated with subcritical water. This observation suggests that in addition to cleavage of the peptide bond, subcritical water treatment results in other hydrolysis products, possibly due to modifications of amino acid side chains. Here, a model peptide comprising all common amino acid residues (VQSIKCADFLHYMENPTWGR) and two further model peptides (VCFQYMDRGDR and VQSIKADFLHYENPTWGR) were treated with subcritical water with the aim of probing any induced amino acid side-chain modifications. The hydrolysis products were analyzed by direct infusion electrospray tandem mass spectrometry, either collision-induced dissociation or electron transfer dissociation, and liquid chromatography collision-induced dissociation tandem mass spectrometry. The results show preferential oxidation of cysteine to sulfinic and sulfonic acid, and oxidation of methionine. In the absence of cysteine and methionine, oxidation of tryptophan was observed. In addition, water loss from aspartic acid and C-terminal amidation were observed in harsher subcritical water conditions.Graphical ᅟ

Highlights

Subcritical water (SCW) exists at a temperature between 100 °C and 374 °C and a pressure less than 22 MPa
In that work we demonstrated that SCW hydrolysis of three standard proteins, hemoglobin, bovine serum albumin (BSA), and beta casein, followed by LC MS/MS of the hydrolysis products, resulted in protein sequence coverages comparable to, or Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users
In order to determine the effects of SCW hydrolysis on the side chains of amino acid residues, a model peptide that incorporates all 20 natural amino acids was designed and synthesized, VQSIKCADFLHYMENPTWGR

Summary

Introduction

Subcritical water (SCW) exists at a temperature between 100 °C and 374 °C and a pressure less than 22 MPa (i.e., below its critical point). CID MS/MS of the species eluting at RT 19 min shows that all three oxidations occur on the cysteine residue, forming sulfonic acid (Supplementary Table 3ii).

Results

Conclusion