This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Protein identification and comprehensive structural characterization of proteins is a major effort using current proteomic methods. Most proteins cannot be identified by mass alone because predicted masses that are based on genomic sequences are not always correct; RNA splicing, protein splicing and Post-Translational Modifications (PTMs) can change the expected protein mass. Therefore other strategies are necessary; the conventional approach follows the bottom-up strategy which is extremely time-consuming as, in most cases gel-separation, followed by proteolysis and then liquid chromatography is required to characterize the protein. This bottom-up approach utilizes the measured masses of proteolysis products and/or uses fragmentation methods to sequence these small proteolysis products to assign protein identities. The major problem is that 100% sequence coverage is extremely unlikely using a single pass proteolysis bottom-up experiment, i.e. often certain parts of the protein sequence are not accounted for because no peptides spanning these regions are observed. Also any peptides that do not match the predicted masses have to be investigated further in order to identify protein modification or sequence variations. Instead of following the bottom-up approach, the alternative, top-down mass spectrometry (MS) can ease this process significantly by eliminating several wet chemistry steps and ensuring complete sequence coverage. The ESI qQq-FTMS instrument built in the laboratory has several advantages for top-down proteomics. Several types of fragmentation are available on this instrument including skimmer fragmentation, Q2 CAD, MSAD, IRMPD, ECD and SORI-CAD - the availability of many different methods of fragmentation facilitates complete sequence coverage as certain motifs and sequences within proteins are more amenable to one method of fragmentation versus another, thus having all the available options increases the possibility of obtaining 100% sequence coverage. In addition, if post-translational modifications are being studied, softer fragmentation methods might be preferable such that the modification is not cleaved from the side chain of the peptide. The fact that this novel instrument has a resolving quadrupole with unit resolution capabilities allows one to use the front-end of the mass spectrometer to isolate the protein of interest from fairly complex mixtures i.e. to separate the ions of the protein of interest from those of background proteins. In addition, in the collision cell allows for the accumulation of the species of interest before transferring it into the ICR cell. This allows top-down studies to be performed on low abundance proteins. We are in the process of optimizing conditions and pulse sequences on this instrument using commercially available proteins such that we have methods in place for the sequencing of proteins from real bioloigal samples. 100% sequence coverage has been obtained for all of these commercially available proteins. In order to perform top- down experiments on samples from human cell lines, methods to isolate intact proteins with modifications have been devised. We have currently devised a biochemical and molecular biological method to isolate complexes such as the Anaphase Promoting Complex and will examine a human complex, the Anaphase Promoting Complex (APC-an E3 ubiquitin ligase) which is known to be highly post-translationally modified during the cell cycle. We have performed top-down experiments on an interaction partner of this complex i.e. UbCH10 (an E2 ubiquitin ligase). The dissection of biological complexes such as the APC have relevance to cancer biology as they are known to control crucial transitions in the cell cycle i.e. the APC initiates anaphase and exit from mitosis.
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