PNNL

Abstract

Single amino acid variants (SAAVs) in protein sequences are often a direct result of single-nucleotide polymorphisms (SNPs). Certain naturally occurring SAAVs have shown biological relevance in different disease conditions but lack precise quantification in circulation which could hinder functional investigations and progress in biomarker development. Here, we have developed a multiplexed liquid chromatography-selected reaction monitoring (LC-SRM) assay that monitors the 9 wildtype-variant peptide pairs (Complement Factor B: CFB-R32Q/R32W, Clusterin: CLU-N317H, Fetuin B: FETUB-K360R, and Kininogen: KNG1-L212P) in non-depleted human plasma. The assay was optimized for imprecision, linearity, stability, and calibration assessments with CVs under 20%. The wildtype-variant peptide pairs were characterized in a set of healthy individual plasma samples. These target identifications were also validated by SNP genotyping with over 99% accuracy. We observed significantly higher concentrations of both CFB variants and a significantly lower concentration of the CLU variant relative to their wild-type (WT) counterparts, while FETUB showed no difference. Furthermore, our results distinguish phenotypes of homozygosity and heterozygosity of SAAV presence through direct concentration level characterization. These findings provide some insights into how SAAVs affects quantitative assessments of target peptides. The assay demonstrates a platform for proteogenomic analyses, with potential applications in both research and clinical settings.