Experimental verification for studying of proteolytic enzymes' primary specificity by statistical analysis of MALDI (matrix-assisted laser desorption/ionization) mass spectra of products obtained by protein substrates proteolysis was done by the use of proteinases with known substrate specificity (trypsin and glutamylendopeptidase). Proposed technique not requires direct determination of proteolysis products amino acid sequences, reliably establishes proteinases with anarrow substrate specificity and shows a relative tolerance for the presence in MALDI mass-spectra peaks of contaminants. It was shown that the pseudo-positive results exception requires the use of protein substrates series with the following averaging received statistical data.
Possibilities and limitations of the method of examination of proteolytic enzymes' primary spec ificity by statistical analysis of MALDI (matrix assisted laser desorption/ionization) mass spectra of products obtained by protein substrate proteolysis, without direct determination of their amino acid sequences, were investigated theoretically. The optimum range given by the measurement errors of the peptides' masses for obtaining a statistical set of the events, and the form of statistical data presentation, were chosen. It was shown that the proposed method can be applied only for proteases with a relatively narrow primary specificity (two or three amino acids). The influence of protein substrate molecular weight and amino acid composition on the efficiency of specifics for a particular protease amino acid, revealed under statistical treatment of the set of proteolysis product masses, was studied on the model of trypsin, chymotrypsin, glutamylendopeptidase, pepsin (pH 1.3).
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