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Targeted Proteomics

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Sample Processing

Two elements unique to the targeted proteomics workflow, an acetone precipitation and short run gel electrophoresis, are included to allow the resource to accommodate as wide a variety of sample sources as possible, to assure a high level of sample consistency, and to provide appropriate QC. The resource can also use in-solution digests for specialized needs.

                               

Data Acquisition 

SRM 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. PRM uses a quadrupole-Orbitrap to perform a full scan of each fragmented peptide target. The primary advantages of the SRM experiments performed on the TSQ Quantiva are the rapid switching between transitions with a set dwell time. The primary advantages of the PRM experiments performed on the Q Exactive Plus are the added specificity of the analysis due to the high product ion m/z resolution and the generally higher sensitivity of the Orbitrap. Most projects will make use of both SRM for panels of high abundance proteins and PRM for panels of lower abundance proteins. PTMs are measured by PRM. The advantage of SRM and PRM is the high specificity of the analysis, but the experiments measure only the predetermined assay targets.  DIA experiments are not biased and can be probed for any target peptide at any time. This targeted probing of DIA data is relatively under-utilized but has a distinct advantage that the targeted processing is based on peptides that have been developed and validated for each protein being measured

 

Assay Development 

We have > 1,000 assays in panels of approx. 75 proteins based on different biochemical pathways of interest. The goal of our assay development process is to identify the two to three peptides that respond best (i.e., the best flyers, etc.). Briefly, a longer list of potential peptides comes from preliminary data and/or consultation with the PeptideAtlas. Peptides are tested experimentally by generating an inclusion list and expected retention times for LC-tandem MS analysis on the quadrupole-Orbitrap instruments in a PRM experiment. The 3 to 5 peptides giving the best response are taken forward. Final choices can be made for the two best flyers based on which peptides give the best chromatographic peak responses and set of product ions to monitor. The process is, however, dependent on having an appropriate sample to analyze. Most of our assays are developed with endogenous proteins in various whole mouse tissue homogenates. The facility also has rat, yeast, worm, and fruit fly samples. Human assays are developed based on different cultured human cells and a small set of human tissue homogenates that have been submitted. Development is independent of final application, so the method can be developed with the sample best suited for that purpose.

Data Processing

We use Skyline to manage our entire process. Briefly, the monitored transitions for each peptide from each protein are built into our array of Skyline methods. Skyline initially finds the iRT peptides and calculates the exact retention times for each target peptide in each run. Chromatograms are reconstructed and peak areas are calculated. These data are manually inspected to assure proper peak identification and integration. The areas are exported and processed to determine geomeans, normalize to the internal standard and calculate pmol/100 ug total protein concentrations using R scripts. QC for all sample groups is evaluated based on the precision for a set of peptides from trypsin autolysis and BSA. Instrument qualification is based on the magnitude of these QC peptides and routine analysis of a digest standard.