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.
Highlights
Subcritical water (SCW) is defined as water maintained at a temperature of between 100 and 374 °C and a pressure of less than 22.064 MPa, i.e., below its critical point
We investigate the specificity of SCW in the hydrolysis of proteins with respect to the production of peptides and compare hydrolysis driven by SCW with enzymatic digestion of proteins by trypsin, the protease of choice for bottom-up proteomics.[18]
Size exclusion chromatography was used to compare peptide size distributions with trypsin resulting in peptides of ∼3500 Da and SCW hydrolysis resulting in a range depending on reaction time
Summary
Subcritical water (SCW) is defined as water maintained at a temperature of between 100 and 374 °C and a pressure of less than 22.064 MPa, i.e., below its critical point. The majority of studies designed to evaluate the utility of the SCW mediated hydrolysis of proteins have focused on the production of amino acids, rather than the intermediate peptides. The yield of amino acids could be increased by increasing both temperature and reaction time. Jung et al showed that pretreatment with SCW resulted in greater efficiency of trypsin digestion of porcine placenta.[19] Marcet et al compared SCW hydrolysis and trypsin digestion for the extraction of peptides from insoluble egg yolk protein.[20] Size exclusion chromatography was used to compare peptide size distributions with trypsin resulting in peptides of ∼3500 Da and SCW hydrolysis resulting in a range (from 1000 Da to 23 kDa) depending on reaction time. High resolution mass spectrometry of superheated ubiquitin confirmed the presence of peptide hydrolysis products
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