Systematic analysis of yeast protein complexes using multidimensional
mass spectrometry.
Jennifer L. Jennings (1), Adrian Canutescu (1), Steven L. Sanders (2),
Melanie D. Ohi (3), Kathleen L. Gould (3), P. Anthony Weil (2),
Andrew J. Link (1)
(1) Microbiology and Immunology, Vanderbilt Univ. Med. School, 1161 21st
ave S, Nashville, TN 37232-2363, USA;
(2) Department of Molecular Physiology & Biophysics;
(3) Department of Cell Biology
One of the most important challenges facing investigators today is to
define the native context in which a given protein functions. It has
become increasingly clear that most proteins work within large
multiprotein complexes. Our goal is to define the native context in
which a given protein functions. Multidimensional chromatography coupled
with tandem mass spectrometry protein identification offers the
potential to rapidly define large multiprotein complexes in an automated
fashion. One such recently developed approach is termed DALPC for Direct
Analysis of Large Protein Complexes. Instead of mass spectrometric
identification of gel-separated proteins, as with conventional mass
spectrometry protein identification, complex protein mixtures are
proteolyzed directly. The resulting peptides are then fractionated by
ion exchange chromatography before reversed-phase HPLC-electrospray
ionization-tandem mass spectrometric identification. In an unbiased
fashion DALPC can identify hundreds to thousands of individual proteins
from complex mixtures in a single analysis without the need for SDS-PAGE
and individual band excision. Statistical and bioinformatics methods are
applied to define the protein interactions and identify
posttranslational modifications. Various approaches to isolate yeast
protein complexes followed by a multidimensional mass spectrometry
analysis will be presented. From our proteomics analysis is emerging a
complex network of protein interactions.
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