Multiplexing Protein Quantification Core

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ABSTRACT The Multiplexed Protein Quantification Core uses LC-tandem MS with selected reaction monitoring (SRM) to measure protein abundance. SRM is unique among the various quantitative mass spectrometry methods because, like Western blots, proteins are carefully selected and targeted for quantification. Targeting is achieved by designing the SRM method to measure the abundance of several peptides, which are unique to the protein when digested with trypsin. These peptides are then detected in complex samples, such as whole tissue homogenates, based on their sequence-specific fragmentation reactions driven by collision- induced dissociation (CID). The peptides are identified as chromatographic peaks at characteristic retention times and the abundance calculated based on the integrated area of those peaks. Two key features of SRM make this a powerful new tool for Geroscience research. First, SRM is a high throughput method where one can measure ~30 proteins in a single assay. The Core has developed panels of protein assays that allow investigators to interrogate entire pathways such as antioxidant proteins, beta oxidation, Krebs cycle, glycolysis, TCA-cycle, and others with new assay panels continuously being developed. Second, a new assay can be designed and validated for any protein from any animal in a few hours. The Core has developed a methodical design process that evaluates the protein sequence using a fundamental understanding of peptide chromatography and gas phase ion chemistry, along with information in public databases, to rapidly select, test, and validate the best peptides for each protein. This rapid and effective assay design/validation process is especially important for aging research using animal models where the availability of antibodies is severely limited. Although SRM has many advantages over immunochemical-based assays like Western blot and ELISA, SRM does require a major piece of equipment (an advanced, high sensitivity LC- tandem mass spectrometry system), expertise to operate that instrument, and expertise for the development and validation of the peptides used to measure each protein. The systems do have the capacity to run as many as 6000 to 8000 samples per year, which makes a core laboratory an excellent way to maximize the benefit of these powerful tools. The Specific Aims for the Targeted Protein Quantitation Core are: Aim 1. Provide high throughput multiplexed protein quantification of panels of proteins in multiple pathways for tissues/cells from mice and rats. Aim 2. Develop new assays and panels for invertebrates and other animal models, e.g., long-lived species, and for proteins requested by individual investigators. Aim 3. Develop the technology to measure the post-translational modifications of proteins.