Absolute Quantification of All Identified Plasma Proteins from SWATH Data for Biomarker Discovery.

The current state of proteomics requires a choice between targeted and global discovery methods. We have established and optimized a method that combines targeted and data-independent acquisition for absolute quantification of all identified plasma proteins in a single Sequential Window Acquisition of all THeoretical fragment ions (SWATH) acquisition run using a panel of Spike-In Standards (SIS). We compared the Absolute Quantification (AQ) of SWATH and Multiple-Reaction Monitoring-High Resolution (MRM-HR) acquisition methods using the 100 protein PlasmaDive SIS panel spiked into non-depleted human plasma. SWATH provided equivalent quantification and differentially abundant protein profiles as MRM-HR. Absolute quantities of the SIS peptides from the SWATH data were used to estimate the absolute quantities (eAQ) for all the proteins in the run. The eAQ values provided similar quantification and differentially abundant protein profiles as AQ and protein area (PA) values. As a proof-of-concept, we applied the eAQ method to 12 plasma samples from 6 non-small cell lung cancer (NSCLC) patients and evaluated the performance of eAQ values versus peak area quantification. There was a strong correlation between AQ and peak area ratios producing significant overlap of differentially abundant proteins. Our eAQ method can provide quantitative data equivalent to AQ or peak area values. This article is protected by copyright. All rights reserved.