Research Interests
Through recent advancements in technology, mass spectrometry–based proteomics is now an important and widespread tool in basic and clinical research. In 2005, VARI purchased a Waters 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 analysis 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 proteins in solutions. Molecular weight determination of protein solutions is typically employed 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 phosphorylation of proteins.
LC-MS Protein Profiling
Liquid chromatography–mass spectrometry is used at most major research institutions to analyze complex protein mixtures for systems biology research and biomarker discovery. 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 at VARI and to those of external clients. Our LC-MS system employs a novel data acquisition method unique to Waters mass spectrometers, termed LC-MSE, 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 the measurement and subsequent comparison of 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 preadipocytes as they differentiate in response to treatment with dexamethasone-insulin or with the PPARγ antagonist rosiglitasone; a second is the study of the secretome of a cell line model of hypoxia. 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 modifications of proteins 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.
As with most mass spectrometry–based methods, mapping phosphorylation sites on proteins begins by enzymatically digesting protein into peptides using trypsin, Lys-C, Staph V8, or chymotrypsin. Peptides are separated by nanoscale reverse-phase HPLC and analyzed by on-line electrospray ionization on a quadrupole time-of-flight (Q-Tof) mass spectrometer. Samples are analyzed using the MSE data acquisition mentioned above. MSE toggles the collision energy in the mass spectrometer between high and low every second throughout the analytic run. Low-collision-energy data acquisition allows peptide mass to be recorded at high sensitivity with high mass accuracy to implicate phosphorylation based on mass alone. The peptide intensity measured in the mass spectrometer is also recorded and used for relative quantitation in time course studies. During high-collision-energy acquisition, all peptides are fragmented to identify the protein(s) from which the peptides were liberated by enzyme digestion and to locate specific phosphorylated amino acids. MSE differs from other mass spectrometry approaches because fragmentation occurs for all peptides, not just for the most abundant peptides. We are currently using this method on several in vitro phosphorylation projects, but our goal is to extend these analyses to in vivo systems to identify novel kinase or phosphatase substrates.
External Collaborators
- Gary Gibson, Henry Ford Hospital, Detroit, Michigan
- Michael Hollingsworth, Eppley Cancer Center, University of Nebraska, Omaha
- Waters Corporation
Core Technology Alliance (CTA)
This laboratory participates in the CTA as a member of the Michigan Proteomics Consortium.
