The OMRF Multiplexing Protein Quantification Core specializes in targeted-validation proteomics. Targeted-validation proteomics typically follows the discovery phase. For targeted-validation proteomics, specialized mass spectrometers are used to specifically quantify levels of a small set of proteins in a large number of samples. Targeted-validation proteomics generally utilizes 1000s of samples to measure 10s of target proteins.
Our analytical workflow has the simple goal of providing as much information to the user as possible within the time and instrument capabilities of the Service Line. We assume the biological samples are precious and difficult/costly to recreate. It is also notable that our sample processing protocol generates sufficient sample for >20 LC-MS experiments. As a result, the resource performs two types of experiments on all samples: (1) targeted quantitative proteomics experiment by SRM and PRM (as dictated by the targets) and (2) an unbiased full scan high resolution experiment as a data archive that can be re-interrogated for new targets, or additional peptides from intriguing results, at any point in the future to answer any potential new questions
The sample preparation pipeline is organized to be robust and reproducible, as well as sufficiently adaptable so as to meet individual project requirements. Each of the specific sample preparation protocols routinely performed in the resource is described below.
SRM and PRM Analysis: Selected reaction monitoring (SRM) analysis relies on the detection of unique “transition” signatures consisting of peptide-ion and fragment-ion pairs on a triple quadrupole instrument to quantify peptides and proteins across sample sets. Parallel reaction monitoring (PRM) analysis uses a quadrupole-Orbitrap instrument to perform a full scan of each fragmented peptide target.
HRAM Analysis: High resolution accurate mass (HRAM) experiments are not biased and can be probed for any target peptide at any time, so the HRAM dataset is a valuable resource with the potential to measure large numbers of additional proteins without the need to repeat the initial biological experiment.
Assay Development: The Service Line currently has approximately 500 assays available for mouse and rat, with additional assays in human, yeast, fruit fly, and worm. We organize assays into panels of approximately 40 assays mostly based on different biochemical pathways. Commonly used panels include glycolysis, gluconeogenesis, Krebs cycle, mitochondrial and peroxisomal beta oxidation, proteostatsis, etc. These assays have been validated over the past 10 years of operation. New assays can be developed by the OMRF site as needed.
Sample processing: A defined amount of protein is taken for analysis, typically 60 µg to 100 µg, with 10 pmol bovine serum albumin added as a non-endogenous internal standard. Samples are taken to 1% SDS, if needed, and heated to break down any sub-cellular compartments and to equilibrate the proteins and internal standard. Finally, the proteins are precipitated with acetone, the sample dissolved in Laemmli buffer at 1 µg/µL, and 20 µg total protein run by short run SDS-PAGE with Coomassie visualization (approx. 1.5 cm into the gel). Each lane is then cut as a sample, reduced, alkylated, and digested with trypsin. The final peptide mixture is extracted from the gel, evaporated to dryness, and reconstituted in 200µL of 1% acetic acid for LC-MS analysis. The resource also uses in-solution digests for specialized needs such as experiments requiring subsequent immunoaffinity purification.
The Targeted Proteomics Service Line is housed at OMRF and operates the below equipment:
Q Exactive Plus
Each instrument is equipped with an Ultimate 3000 split-less nanflow HPLC with autosampler.
Contact the OMRF site for any inquiries regarding instrumentation.