Combining peptide modeling and capillary electrophoresis-mass spectrometry for characterization of enzymes cleavage patterns: recombinant versus natural bovine pepsin A.
Nowadays there is an increasing number of recombinant enzymes made available to industry. Before replacing the use of natural enzymes with their cognate recombinant counterparts, one important issue to address is their actual equivalence. For a given recombinant proteolytic enzyme, its equivalence can be investigated by comparing its cleavage specificity with that obtained from the natural enzyme. This is mostly done by analyzing the fragments (i.e., peptidic map) attained after enzymatic digestion of a given protein used as substrate. The peptidic maps obtained are typically characterized using separation techniques together with MS and MS/MS systems. However, these procedures are known to be difficult and labor-intensive. In this work, the combined use of a theoretical model that relates electrophoretic behavior of peptides to their sequence together with capillary electrophoresis-mass spectrometry (CE-MS) is proposed to characterize in a very fast and simple way the cleavage specificity of new recombinant enzymes. Namely, the effectiveness of this procedure is demonstrated by analyzing in few minutes the fragments obtained from a protein hydrolysated using recombinant and natural pepsin A. The usefulness of this strategy is further corroborated by CE-MS/MS. The proposed procedure is applicable in many other proteomic studies involving CE-MS of peptides.
Simó
Carolina
C
Institute of Industrial Fermentations (CSIC), Juan de la Cierva 3, Madrid, Spain.
González
Ramón
R
Barbas
Coral
C
Cifuentes
Alejandro
A
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods*; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results
D010446:Peptide Fragments; D011994:Recombinant Proteins; D010434:Pepsin A