Plans: The specific objectives of project 4 over the next 12 months are to concentrate on developing more methods of protein separation, quantifying the levels of protein and identifying them by mass spectrometry. There are many reasons for the continued methods development, to: prepare tissues properly for subsequent mass spectral analyses; devise alternative ways to confirm and expand initial mass spectral findings; and have available a variety of strategies that provide enough purified protein for situations in which the initial LC-MS\/MS is not sufficient or sequence coverage of the MALDI spectra of a recombinant protein, GPx-1 for example, is not adequate. Project 4 investigators have results from a liver proteomics scan of hyper-homocysteinemic and wildtype mice performed in the previous year and want to develop further methods of protein separation and quantification to confirm and expand initial MS findings.<\/p>\n
Investigators progressed in the use of a variety of separation methods for analysis of protein abundance and modifications. 1) Samples prepared from liver of hyper-homocysteinemic and wildtype mice were tested using Western blots to assess integrity for further analysis by a protein fractionation 2-D chromatography method. Preliminary Western blots indicate intact proteins. Samples are stored at -80 \u00b0C pending separation by the PF2D system. 2) A column-based approach was used to isolate additional recombinant GPx-1 protein with the goal of obtaining spectrum for the selenocysteine-containing peptide in untreated samples. After gel purification and trypsin digestion, researchers observed a peak in several MALDI-TOF mass spectra whose m\/z value was consistent with the [M+H]+ expected for the selenocysteine-modified peptide . They isolated more of the sample to localize the selenocysteine peak by LC-MS\/MS but to date have not found selenocysteine-containing peaks by this method. 3) A novel in situ assay to assess disulfide bond formation in proteins in fixed cells was developed. The formation of disulfide bonds appears to be regulated by hydrogen peroxide levels. Since GPx-1 is responsible for eliminating cellular peroxides, researchers are assessing whether alterations in GPx-1 expression levels affect disulfide bond formation. Following up previous studies, they prepared a recombinant GPx-1 construct without a His-tag, tested the effectiveness of the construct to overexpress GPx-1 in transiently transfected cells, and started the process of selecting for permanent transfectants.<\/p>\n","protected":false},"excerpt":{"rendered":"
Plans: The specific objectives of project 4 over the next 12 months are to concentrate on developing more methods of protein separation, quantifying the levels of protein and identifying them by mass spectrometry. There are many reasons for the continued methods development, to: prepare tissues properly for subsequent mass spectral analyses; devise alternative ways to […]<\/p>\n","protected":false},"author":2,"featured_media":0,"parent":0,"menu_order":77,"comment_status":"closed","ping_status":"closed","template":"","meta":[],"_links":{"self":[{"href":"https:\/\/www.bumc.bu.edu\/cardiovascularproteomics\/wp-json\/wp\/v2\/pages\/57"}],"collection":[{"href":"https:\/\/www.bumc.bu.edu\/cardiovascularproteomics\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.bumc.bu.edu\/cardiovascularproteomics\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.bumc.bu.edu\/cardiovascularproteomics\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/www.bumc.bu.edu\/cardiovascularproteomics\/wp-json\/wp\/v2\/comments?post=57"}],"version-history":[{"count":7,"href":"https:\/\/www.bumc.bu.edu\/cardiovascularproteomics\/wp-json\/wp\/v2\/pages\/57\/revisions"}],"predecessor-version":[{"id":1804,"href":"https:\/\/www.bumc.bu.edu\/cardiovascularproteomics\/wp-json\/wp\/v2\/pages\/57\/revisions\/1804"}],"wp:attachment":[{"href":"https:\/\/www.bumc.bu.edu\/cardiovascularproteomics\/wp-json\/wp\/v2\/media?parent=57"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}