Analysis of proteoforms is the next challenging level in proteomics (1). Identification of proteoforms in relationship to their function is demanding. Genes coding proteases usually have different proteoforms, including inactive proteoforms, which in many cases are converted by a proteolytic truncation into active proteoform. Active proteases are controlled by protease inhibitors. Some of these inhibitors belonging to the suicide inhibitors are binding covalently to the active proteoform, thereby forming a new inhibited proteoform - a fusion protein consisting of the protease and the inhibitor.
We have developed a method by which proteoforms, coded by a single gene, with different functional status can be identified (2). Therefore, the sample containing different proteofoms is separated with respect to the molecular weights of the proteins. The proteins of each of the resulting fractions are digested with trypsin. The tryptic peptides from each of the different fractions are analyzed by LC-MSMS, the peptides identified by a search engine, the relative amounts calculated by the area under the curves (AUCs) of their extracted ion chromatograms (EICs) and these values plotted against the molecular weight of the fractions. Thereby, proteoforms coded by the same gene but with different molecular weight can be identified. We applied this approach for analyzing different functional status of proteoforms of factor XII (FXII) in human blood plasma, including the inactive precursor, the activated proteoform generated by proteolysis, and several proteoforms inhibited by different inhibitors like the C1-inhibitor (C1-Inh). Furthermore this approach is giving access to the identification of substrates and their proteoforms (here: e.g. kallikrein) of the target protease as well as down-stream proteases and substrates (here: e.g. kininogen) of proteolyse products.
In summary, we have developed a tool, by which the functional status of protease proteoforms and their interactomes as well as corresponding protease cascades can be investigated.