Targeted proteomic assays for quantitation of proteins identified by proteogenomic analysis of ovarian cancer

Abstract

Mass spectrometry (MS) based targeted proteomic methods such as selected reaction monitoring (SRM) are emerging as a promising tool for verification of candidate proteins in biological and biomedical applications. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute has investigated the standardization and analytical validation of the SRM assays and demonstrated robust analytical performance on different instruments across different laboratories. An Assay Portal has also been established by CPTAC to provide the research community a resource consisting of large sets of targeted MS-based assays, and a depository to share assays publicly. Herein, we report the development of 98 SRM assays that have been thoroughly characterized according to the CPTAC Assay Characterization Guidance Document; 37 of these passed all five experimental tests. The assays cover 70 proteins previously identified at the protein level in ovarian tumors. The experiments, methods and results for characterizing these SRM assays for their MS response, repeatability, selectivity, stability, and endogenous detection are described in detail. Data are available via PeptideAtlas, Panorama and the CPTAC Assay Portal.

Figure 1. Summary of the five experiments for characterizing the targeted MS assays as recommended by the CPTAC Assay Portal.

Experiments 1 and 2 are required for uploading the assays to the Assay Portal while Experiments 3 to 5 are optional. (a) Experiment 1 is designated to create a response curve using multiple points of various concentrations of the target peptides. (b) Experiment 2 determines the repeatability of assays at 3 different concentration points over 5 days. This mini-validation evaluates inter- and intra-day variability. (c) Experiment 3 studies the selectivity (parallelism) of the assays in six different biological replicates of the same matrix type. (d) Experiment 4 is designed to check the stability of the target peptides at different storage time and conditions. (e) Experiment 5 applies the overall workflow developed in the previous experiments to the detection of the endogenous peptides in the biological samples.

Figure 2. An example of the evaluation results from the five experiments.

The data was associated with peptide FSASGELGNGNIK from the proliferating cell nuclear antigen (PCNA) protein. (a) A representation of extracted ion chromatograms for light and heavy peptides each with three transitions. (b) Peak area ratio of light to heavy peptides for each transition and summation of transitions (sum) was used to plot the linear slope over different concentration points with representative R² values. An inset graph shows the linear slope over the low range of concentration points. (c) The measurement variability of this assay was assessed at 3 different concentration points across 5 days. (d) Similar to Experiment 1, the linear slope was constructed at three concentration points to assess the influence of matrix differences. It was performed for 6 different biological matrixes (M1 to M6) with representative R2 values. (e) From Experiment 4, the stability of heavy peptides can be determined by creating the peak area ratio for each transition and their summed value. This can help the researchers to determine proper handling of peptides. (f) When the endogenous peptides were detected, peak area ratios of endogenous to heavy peptides were represented for each transition and their summed value. The reproducible detection can be checked using the 5 full process replicates from the sample preparation to LC-SRM analysis.