Implementation of Multidimensional Protein Identification Technology and its Application to the Characterization of Protein Complexes in Bakers Yeast

mass spectrometry;protein complexes;Proteomics

Graumann, Johannes ; Deshaies, Raymond Joseph

University:California Institute of Technology

Department:Biology

关键词: mass spectrometry;    protein complexes;    Proteomics;   

Others  :  https://thesis.library.caltech.edu/2103/2/Graumann_PhD-Thesis.pdf

美国|英语

来源: Caltech THESIS

【 摘 要 】

The analysis of complex polypeptide mixtures poses a central and ubiquitous problem to biochemistry, molecular and cellular biology. Historically the problem has been approached by means of gel electrophoretic separation, coupled to immune-chemistry or Edman degradation (Edman 1949) based identification of separated components. These approaches as well as those based on liquid chromatography are hampered by a central issue: the wide spectrum of polypeptide characteristics that renders their separation difficult. A recent strategy termed multidimensional protein identification technology (MudPIT) tackles this problem by capillary chromatographic separation of not the complete polypeptides, but rather peptides yielded by them through proteolytic digest and analyzing them in-line using ion trap mass spectrometry (Link et al. 1999; Washburn et al. 2001; Wolters et al. 2001).

This work describes the implementation of MudPIT outside of the analytical chemistry environment of its inception. Robustness and generalizability of the technique are tested by analysis of polypeptide complexes copurifyed with 25 selected gene products from Saccharomyces cerevisiae (Graumann et al. 2004). The pilot study reveals MudPIT to be mature enough for use outside of specialized environments and, by yielding with Rtt102p a novel component of the Swi/Snf and RSC chromatin remodelling complexes, to have potential for delivering new insights even into extensively studied systems.

Subsequent application of MudPIT to the characterization of components of the ubiquitin-proteasome system (Verma et al. 2004; Mayor et al. 2005) and mitochondrial fission (Griffin et al. 2005) in S. cerevisiae further emphasize its potential to contribute to biochemical research.