A Multiplexed Quantitative Analysis of Germline Single Amino Acid Variants by Targeted Proteomics in Nondepleted Human Plasma

Single amino acid variants (SAAVs) in protein sequences are often a direct result of single-nucleotide polymorphisms (SNPs). Certain germline 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 5 wild-type and variant peptide pairs (Complement Factor B: CFB-R32Q/R32W, Clusterin: CLU-N317H, Fetuin B: FETUB-K360R, and Kininogen: KNG1-L212P) in nondepleted human plasma. The assay was optimized for imprecision, linearity, stability, and calibration assessments with CVs of under 20%. The wild-type and variant peptide pairs were characterized in a set of healthy individual plasma samples. These target identifications were also validated by SNP genotyping with more than 99% accuracy. For all protein targets, we observed significantly lower concentrations of WT species in the presence variant peptides. In CFB, the concentration of R32Q was significantly lower than its counterpart R32W variant and WT species. Furthermore, our results distinguished phenotypes of homozygosity and heterozygosity of the SAAV presence through direct concentration level characterization. These findings provide some insights into how SAAVs affect quantitative assessments of target peptides. The assay demonstrates a platform for proteogenomic analyses with potential applications in both research and clinical settings.蛋白质序列中的单氨基酸变异体 (Single Amino Acid Variants, SAAVs) 通常由单核苷酸多态性 (Single-Nucleotide Polymorphisms, SNPs) 直接引发。部分生殖系SAAVs在多种疾病状态下已被证实具有生物学相关性，但目前尚缺乏对其循环水平的精准定量检测手段，这可能会阻碍功能研究与生物标志物开发的进展。本研究开发了一种多重液相色谱-选择反应监测 (Liquid Chromatography-Selected Reaction Monitoring, LC-SRM) 检测方法，可在未经蛋白耗竭的人血浆中定量5组野生型与变异型肽对：补体因子B (Complement Factor B, CFB)-R32Q/R32W、簇集蛋白 (Clusterin, CLU)-N317H、胎球蛋白B (Fetuin B, FETUB)-K360R以及激肽原 (Kininogen, KNG1)-L212P。该检测方法针对不精密度、线性范围、稳定性以及校准验证进行了优化，其变异系数 (Coefficient of Variation, CV) 均低于20%。研究在一批健康个体血浆样本中对上述野生型与变异型肽对进行了表征分析，上述靶点的鉴定结果也通过SNP基因分型得到了验证，准确率超过99%。在所有蛋白靶点中，当存在变异型肽时，我们均观测到野生型 (Wild-Type, WT) 肽的浓度显著降低。在CFB靶点中，R32Q变异型肽的浓度显著低于其同源变异型R32W以及野生型肽。此外，本研究通过直接定量浓度表征，区分了存在SAAVs的纯合子与杂合子表型。上述研究结果为阐明SAAVs如何影响靶点肽的定量评估提供了新的见解。该检测方法构建了一个蛋白质基因组学分析平台，在科研与临床场景中均具有潜在应用价值。