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。部分生殖系单氨基酸变异体在多种疾病状态下已被证实具有生物学相关性，但在循环系统中缺乏精准定量数据，这可能会阻碍功能研究及生物标志物开发进程。本研究开发了一种多重液相色谱-选择反应监测（LC-SRM）检测方法，可在未耗竭的人血浆中对5组野生型与变异肽对进行定量检测：补体因子B（Complement Factor B, CFB）的CFB-R32Q/R32W、簇蛋白（Clusterin, CLU）的CLU-N317H、胎球蛋白B（Fetuin B, FETUB）的FETUB-K360R以及激肽原（Kininogen, KNG1）的KNG1-L212P。该检测方法针对不精密度、线性、稳定性及校准性能进行了优化，其变异系数（coefficient of variation, CV）低于20%。我们在一组健康个体血浆样本中对上述野生型与变异肽对进行了表征，并通过SNP基因分型验证了靶点鉴定结果，准确率达99%以上。对于所有靶蛋白，我们观察到：当存在变异肽时，野生型肽的浓度显著降低。在补体因子B中，R32Q变异体的浓度显著低于其同源变异体R32W及野生型肽。此外，本研究通过直接浓度水平表征，区分了单氨基酸变异体存在的纯合子与杂合子表型。这些发现为理解单氨基酸变异体如何影响靶肽的定量分析提供了新思路。该检测方法构建了蛋白质基因组学分析平台，有望在科研与临床场景中得到应用。