Research Core Services
Proteomics and Metabolomics Laboratory
It is essential to provide accurate, reliable, and consistent data in analytical services. Based on the need of investigators, we provide services for developing analytical methods using a) HPLC-UV, b) HPLC-Fluorescence, and c) LC/MS/MS for analysis of endogenous compounds and xenobiotics in biological matrices like plasma, urine and tissues.
Novel Compound Identification
Investigators interested in isolating compounds from biological sources should, establish the activity of compound, measure the molecular weight, and determine the compound structure. We provide services using HPLC for purification of the compound of interest and LC/MS/MS for identifying the molecular weight of the compound and for producing structure confirmation if needed.
Small Molecule Quantitation
Small molecules (100-800 Daltons) have a variety of biological functions, serving as cell signaling molecules, as tools in molecular biology, and as drugs in medicine. Liquid chromatography on-line tandem mass spectrometry (LC/MS/MS) is the method of choice for small molecule quantitation because it provides accurate, reliable and consistent data and requires fewer specimens. We provide services using LC/MS/MS for quantitation of small molecules in complex biological matrices, such as plasma, urine and cell extracts.
Isotope Enrichment analysis
These experiments allow the utilization of LC-MS for the determination of protein turnover rates. The method that has been developed involves the incorporation of a heavy isotope, 2H, into proteins by the administration of 2H2O to animal models of disease. These experiments require several time points for the measurement of peptide isotope enrichment at each time point which allows the calculation of protein synthesis or degradation rates.
Global Phosphoproteomic analysis
Global phosphoproteomic experiments attempt to identify and/or quantify protein phosphorylation on a proteome wide scale. Protein phosphorylation is one of the most common post-translational modifications and is involved in many cellular processes including cellular signaling, cell survival, regulation of transcription and translation, proliferation, and differentiation. Due to the complexity of the cellular and tissue proteomes along with the oftentimes low stoichiometry of the phosphorylation events, phosphopeptide enrichment has to be utilized. This service includes protein digestion, phosphopeptide enrichment, LC-MS/MS analysis, and data interpretation.
Molecular Weight Analysis
The analytical method used for molecular weight analysis is LC-MS and can be used to analyze peptides or proteins which are less than 30 kDa in size.
These experiments are usually performed in a protein specific manner. The protein is digested with multiple proteases to increase overall sequence coverage and these digests are analyzed looking for specific post-translational modifications. Some of the modifications that have been identified by the core include phosphorylation, methylation, acetylation, ubiquitination, and sulfation. These experiments can be performed from either an in-gel or in-solution protein digestion, however, gel isolated samples are preferred.
These experiments involve the digestion of proteins with trypsin followed by LC-MS analysis. The complexity of the samples that the core can analyze can vary between samples containing only a single protein to samples with 100s to 1000s of proteins. The core can perform protein identification from either gel bands or from proteins in solution.
The LC-MS method used by the Proteomics core is inherently quantitative, and the Proteomics Core can perform quantitative analysis on proteins and post-translational modifications. These quantitative experiments are performed using either label free methods, such as spectral counting or Selective Reaction Monitoring, or methods that include the incorporation of stable isotopes. The stable isotope methods that are currently supported by the Core include SILAC and isobaric labeling.