Design of a LC-MS/MS Plasma Protein Assay Using Data Independent Acquisition
University of Washington
A data independent acquisition (DIA) approach is used to generate a highly multiplexed plasma protein assay. Using DIA, we build a chromatogram library containing relative fragment ion abundances and retention times for thousands of peptides detected in human plasma. Significantly, we use the high multiplexing capacity of DIA to expedite assay validation including assessment of digestion kinetics, peptide stability, and quantitative linearity. Additionally, we demonstrate an external calibration approach for DIA-based assays using a pooled plasma standard. The plasma standard is measured intermittently using a single LC-MS/MS DIA injection. The signal for each peptide measurement can then be calibrated to the signal for the same peptide in the plasma standard to control for batch effects and normalize to a common reference.
Presented at MSACL 2016 US