Evaluation of PQ500, a 500-plasma protein blood panel in NSCLC subjects using high-throughput MRM mass spectrometry.

Abstract

110 Background: Monitoring of circulating proteins in cancer subjects can provide insight into the host response to the tumor. Recent studies have demonstrated the utility of circulating proteins for early detection, differential diagnosis, and predicting response to therapy. Although the utility is clear, standardization of measurement methods across a large number of proteins from plasma has remained challenging. Here, we evaluate the detection of proteins from a > 500-protein plasma panel assembled from stable-isotope standard (SIS) peptides and measured in a high-throughput MRM workflow. Methods: Plasma samples from subjects with Stage III-IV non-small cell lung cancer (n = 15), Stage IV pancreatic cancer (n = 6) and healthy donors (n = 15) were prepared for analysis. Prior to analysis, a panel of SIS peptides covering 582 plasma proteins (804 peptides), was spiked into each sample enabling absolute quantification of detectable peptide species. All samples were analyzed using a capillary flow liquid chromatography (5 µl/min) with a one-hour gradient coupled to a Thermo Scientific TSQ Altis triple quadrupole mass spectrometer. Protein data was extracted and quantified for statistical analysis using SpectroDive (Biognosys). Results: Across all samples, 607 peptides representing 411 proteins were quantified at least once, with an average of 300 proteins quantified per sample. From triplicate analyses, the median CV across all proteins was 5.3%. Statistical testing identified 42 significantly regulated proteins (36 up- and 6 down-regulated; q-value > 0.05 and log2 fold change > 0.58). Key species that were strongly dysregulated in cancer samples included: c-reactive protein (CRP), serum amyloid a (SAA1/SAA2), complement C9, S100A8/S100A9, and leucine rich glycoprotein 1 (LRG1). Conclusions: PQ500 is a comprehensive blood-focused multiplexed biomarker panel that enables absolute quantification of plasma proteins. The dysregulated proteins identified in this study highlight that the PQ500 can monitor the host immune response through multiple pathways. Accurate monitoring these proteins offers the possibility to define molecular based markers with clinical and diagnostic utility.