Subcritical Water Processing of Proteins: An Alternative to Enzymatic Digestion?

Abstract

Subcritical water is an emerging tool in the processing of bioorganic waste. Subcritical water is an environmentally benign solvent which has the potential to provide an alternative to traditional methods of protein hydrolysis without the inclusion of expensive acids or enzymes. To date, most studies on the subcritical water mediated hydrolysis of proteins have focused on the production of amino acids, rather than the intermediate peptides. Here, we investigate the specificity of subcritical water with respect to the production of peptides from three model proteins, hemoglobin, bovine serum albumin, and β-casein, and compare the results with enzymatic digestion of proteins by trypsin. In addition, the effect of subcritical water (SCW) treatment on two protein post-translational modifications, disulfide bonds and phosphorylation, was investigated. The results show that high protein sequence coverages (>80%) can be obtained following subcritical water hydrolysis. These are comparable to those obtained following treatment with tryspin. Under mild subcritical water conditions (160 °C), all proteins showed favored cleavage of the Asp-X bond. The results for β-casein revealed favored cleavage of the Glu-X bond at subcritical water temperatures of 160 and 207 °C. That was similarly observed for bovine serum albumin at a subcritical water temperature of 207 °C. Subcritical water treatment results in very limited cleavage of disulfide bonds. Reduction and alkylation of proteins either prior to or post subcritical water treatment improve reported protein sequence coverages. The results for phosphoprotein β-casein show that, under mild subcritical water conditions, phosphorylation may be retained on the peptide hydrolysis products.

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

“…This assignment was confirmed by CID (data not shown). The peptide product is the result of cleavage C-terminal to the aspartic acid residue in the original peptide, consistent with our previous work, which found aspartic acid to be the most common site of SCW-induced cleavage [ 2 ]. Oxidation of this peptide product was also observed at m / z 522.9094 ( m / z calc 522.9105).…”

“…In each case, the residency time was 10 min. A greater amount of peptide bond hydrolysis was observed as the temperature increased, as expected from our earlier work [ 2 ]. Following treatment at 160 °C, the cleavage product FLHYMENPTWGR represents the base peak in the mass spectrum.…”

“…An arginine residue was placed at the C-terminus of the peptide in order to allow efficient generation of a ‘y’ or ‘z’ fragment ion series following fragmentation. The acidic glutamate and aspartate residues, which are known to direct backbone cleavage in SCW conditions [ 2 ], were separated by five amino acid residues. The direct infusion electrospray mass spectrum of the peptide is shown in Supplementary Figure 1 , with peak assignments detailed in Supplementary Table 1 .…”

“…In light of the above results, the data obtained from the SCW treatment of proteins in our original publication [ 2 ] were re-analyzed. The data were searched against the relevant protein sequence as obtained from UniProt using the SEQUEST algorithm in Proteome Discoverer ver.…”

“…In the subcritical state, the ionic product of water, K w , increases with temperature, resulting in the formation of hydronium (H 3 O + ) and hydroxide (OH – ) ions, enabling SCW to act as either an acid or base catalyst [ 1 ]. We have previously investigated SCW as an alternative to tryptic digestion in the bottom-up analysis of proteins [ 2 ]. 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 greater than, that of trypsin.…”