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Mass spectrometry has gained prominence in limited proteolysis studies largely due to its unparalleled precision in determining protein molecular mass. However, proteolytic fragments usually cannot be identified through direct mass measurement, since multiple subsequences of a protein can frequently be matched to observed masses of proteolytic fragments. Therefore, additional information from N-terminal sequencing is often needed. Here we demonstrate that mass spectrometry analysis of the time course of limited proteolysis reactions provides new information that is self-sufficient to identify all proteolytic fragments. The method uses a non-specific protease like subtilisin and exploits information contained in the time-resolved dataset such as: increased likelihood of identifying larger fragments generated during initial proteolysis solely by their masses, additivity of the masses of two mutually exclusive sequence regions that generate the full-length molecule (or an already assigned subfragment), and analyses of the proteolytic subfragment patterns that are facilitated by having established the initial cleavage sites. We show that the identities of the observed proteolytic fragments can be determined by LC/MS alone because enough constraints exist in the time-resolved dataset. For a medium-sized protein, it takes about 8 h to complete the study, a significant improvement over the traditional SDS-PAGE and N-terminal sequencing method, which usually takes several days. We illustrate this method with application to the catalytic domain of mitogen-activated protein kinase-activated protein kinase-2, and compare the results with N-terminal sequencing data and the known X-ray crystal structure.

Time-resolved limited proteolysis of mitogen-activated protein kinase-activated protein kinase-2 determined by LC/MS only