Characterization of 53 Multiplexed Targeted Proteomics Assays for Verification Studies in Cancer Cell Lines

The National Cancer Institute’s Clinical Proteomics
Tumor
Analysis Consortium (CPTAC) was established to address the need for
improved design, standardization, and validation of proteomics assays
to enable better translation of biomarkers from the analytical lab
to the clinic. Here, we applied CPTAC guidelines to characterize quantitative
mass spectrometry (MS) assays in a new multiple reaction monitoring
(MRM) proteomics panel. The panel of 50 proteins was developed in
response to a previous study that identified a proteomic profile of
altered translational control associated with response to a new cancer
drug. MRM-MS assays for 53 peptides of interest were developed, optimized,
and characterized on a UPLC system coupled to a triple-quadrupole
mass spectrometer (QQQ-MS) using synthetic proteotypic peptides and
corresponding stable-isotope labeled internal standard (SIS) peptides.
Most of the assays were found to be fit-for-purpose for biomarker
verification in that they precisely and reproducibly quantify the
peptides at levels corresponding to the endogenous concentration in
the desired cancer cell lines. Of these, 28 peptide assays represent
to proteins that previously had no associated assays published in
the CPTAC database. The targeted proteins in this publicly deposited
validated multiplexed panel may be of use for research applications
in cancer, cellular stress, neurology, cardiology, and metabolism.