KCS

Although one of the most important and established roles of top-down protein analysis is localization of protein modifications, there does exist a nascent need for identification from these analyses to complement shotgun type ID’s performed via bottom-up approaches. We present initial results of a new version of MASCOT (BigMASCOT) that has been recompiled to allow searches of proteins up to 80 kDa, enabling it for the first time to be used as a tool for top-down identification. In addition to showing BigMASCOT processing results on a variety of mass spec standards, we present results from phosphorylated and truncated human proteins correctly identified without any prior knowledge of the protein state. To demonstrate this concept, we present results from a series of variants of Human SOD-1, UHCL-1 and DJ-1, many of which differ from the unmodified sequence only by a point mutation or deletion. These proteins were introduced into the 9.4T apex-Qe where the intact molecular weight was measured followed by fragmentation with ECD or in-source CAD. Processing this data with bigMASCOT yielded the correct identification every time for proteins matched against a special version of the MSDB that contained all of the SOD-1 variants.