Scientific Overview: Mass Spectrometry and Proteomics

Research Interests

The Mass Spectrometry and Proteomics laboratory provides protein identification analysis and protein molecular weight determination as core services. Nanogram amounts of protein in SDS-PAGE gels or in solution are digested into peptides and analyzed by HPLC with on-line electrospray mass spectrometry. Peptides are fragmented in the mass spectrometer to generate amino acid sequence data that is used to identify proteins by searching protein and DNA databases. Submicrogram amounts of intact proteins are analyzed by nanoscale liquid chromatography–mass spectrometry (LC-MS) to determine their average molecular weight; this work is performed using a variety of HPLC columns to optimize recovery and provide reliable results. These core services are provided to both VARI investigators and external clients. Research in the lab focuses on improving existing services and developing new methods based on the needs of VARI investigators. Our three main areas of interest are intact-protein molecular weight determination, phosphopeptide analysis, and protein expression profiling using LC-MS.

Protein LC-MS

We use protein LC-MS to confirm correct expression and purification of recombinant proteins from bacteria. The average molecular weight of a protein is experimentally determined and compared with the calculated weight from the expected amino acid sequence. Proteins of 50 kDa and larger are analyzed with mass accuracy often better than 0.01%, or ±1 Da per 10 kDa. Unlike with conventional SDS-PAGE, protein truncation and modifications such as oxidation or acetylation can be accurately characterized using protein LC-MS. This information is essential when protein reagents are used for labor-intensive and costly protocols such as x-ray crystallography, antibody production, or drug screening. We have a dedicated LC-MS instrument with optimized HPLC separation and comprehensive data processing for analyzing complex mixtures of proteins. For proteins that degrade during purification, we can alter the use of protease inhibitors or minimize degradation through site-directed mutagenesis of susceptible amino acids. We are also exploring the use of this equipment for biomarker discovery of intact proteins. The goal is to provide relative quantitation of proteins in disease cell culture models, tumor tissue, and cancer patient body fluids.

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 potential drug targets in cancer 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. Our lab collaborates with investigators to map protein phosphorylation using techniques including multiple enzyme digestion, titanium dioxide phosphopeptide enrichment, and phosphorylation-specific mass spectrometry detection. Although trypsin is often the enzyme of choice for digesting proteins into peptides for identification, additional enzymes such as Lys-C, Staph V8, chymotrypsin, thermolysin, or elastase may also be employed. Multiple enzyme digests and titanium dioxide enrichment are used in combination with precursor ion scanning for –79 m/z on a Waters Q-Tof Premier mass spectrometer.

We have developed a robust negative-ion-mode method using nanoscale HPLC that provides specific detection of phosphopeptides below 20 fmol in the presence of 2 pmol of nonphosphorylated protein. Once detected in the negative mode, phosphopeptides are sequenced in a subsequent LC-MS analysis in the positive ion mode using accurate mass parent ion selection, a narrow retention time window, and collision energy ramping. This approach has provided a reliable and sensitive means of analyzing phosphoproteins in our laboratory. Our current focus is on applying this label-free method to studies requiring relative quantitation of phosphorylation events.

Protein expression/biomarker discovery

As mass spectrometry instruments and protein separation methods develop, proteomics techniques allow researchers to identify and quantitate protein samples of increasing complexity. The ultimate goal is to catalog all proteins expressed in a given cell or tissue as a means of evaluating dynamic physiological events and understanding how all proteins interact to affect a biological outcome. Traditionally this goal has been approached using 2D gel electrophoresis, image analysis of stained proteins, and identification of proteins from gels using mass spectrometry. Because of the labor-intensive nature of 2D gels and the underrepresentation of some protein classes (such as membrane proteins), proteomics has been moving toward solution-based separations and direct mass spectrometry analysis. Our laboratory recently purchased and installed a Waters Corporation Protein Expression System for non-gel-based, label-free protein expression analysis. This system represents a paradigm shift in the field of proteomics, because it provides both quantitative and qualitative data on complex mixtures of proteins in a single LC-MS analysis. Proteins are enzymatically digested using trypsin and, without any chemical or isotopic labeling, the resulting peptides are analyzed by LC-MS. The combination of molecular mass and LC retention time establishes a signature for each peptide and allows comparison across samples. The mass spectrometer signal intensity of each peptide is used for quantitation. Qualitative protein identification data is obtained by fragmenting all peptides eluting into the mass spectrometer, a feature unique to the Waters instrument. VARI is one of an elite group of institutions that have this powerful new technology. This system will be used to map protein pathways under a systems biology approach and to discover potential biomarkers for early detection and diagnosis in cancer and other diseases.

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 (MPC).