2009 Scientific Report

VARI | 2009
Research Interests
Mass spectrometry–based proteomics is now an important and widespread tool in basic and clinical research. In 2005, VARI
purchased a Waters quadrupole time-of-flight (Q-Tof) mass spectrometry system that remains at the cutting edge of many
research applications. This equipment allows us to provide routine mass spectrometry services and to develop new services
such as protein profiling for biomarker discovery and protein phosphorylation analysis.
Protein identification and protein molecular weight determination are routine services performed on sub-microgram amounts
of material to address a wide variety of biological questions. Protein identification via mass spectrometry is mainly used to
identify novel protein-protein interactions and can be performed on proteins in SDS-PAGE gels or in solutions. Molecular
weight determination of protein solutions is typically used to confirm the expression and purification of recombinant proteins to
be used as reagents in x-ray crystallographic experiments or drug screening/cell-based assays. Our research emphasis is on
1) developing liquid chromatography–mass spectrometry (LC-MS) protein profiling analysis for systems biology research and
biomarker discovery and 2) improving methods for identifying and quantifying the phosphorylation of proteins.
LC-MS protein profiling
Our lab collaborates with Waters Corporation, a major manufacturer of mass spectrometry and HPLC equipment, to evaluate
and improve existing methods while applying LC-MS to the research efforts of VARI scientists and of external clients. Our
LC-MS system employs a novel data acquisition method unique to Waters mass spectrometers, termed LC-MS E , whereby
quantitative and qualitative data are collected in a single analysis. Protein samples are first digested into peptides using
trypsin and then analyzed by reverse-phase nanoscale LC-MS. Recording peptide mass, HPLC retention time, and intensity
as measured in the mass spectrometer, we digitize the data to allow comparisons across samples. Quantitation is based on
measuring and comparing the chromatographic peak area for each peptide across samples. Qualitative protein identification
data is collected in a multiplexed, non-intensity-biased fashion concurrent with quantitative data. One current pilot project is a
time-course analysis of protein secretion (secretome) from mouse 3T3-L1 pre-adipocytes as they differentiate in response to
treatment with dexamethasone/insulin, versus the response to the PPARg antagonist rosiglitazone. A second study is of the
secretome of a cell line model of cachexia.
In addition to mechanism-of-action studies, our goal is to use LC-MS to discover candidate biomarkers of disease. Current
research efforts focus on sample processing techniques to reproducibly fractionate highly complex samples such as blood
plasma, tissue, and urine to allow quantitative analysis. Replicate LC-MS analysis of carefully chosen samples and multivariate
data analysis will allow us to differentiate between normal biological variation and disease.
Protein phosphorylation analysis
Mapping post-translational protein modifications such as phosphorylation is an important yet difficult undertaking. In cancer
research, phosphorylation regulates many protein pathways that could serve as targets for drug therapy. In recent years,
mass spectrometry has emerged as a primary tool in determining site-specific phosphorylation and relative quantitation.
Phosphorylation analysis is complicated by many factors, but principally by the low-stoichiometry modifications that may
regulate pathways: we are sometimes dealing with 0.01% or less of phosphorylated protein among a large excess of a
nonphosphorylated counterpart.
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