A targeted, bottom-up proteomic assay was developed for the qualitative detection of apolipoprotein L1 (ApoL1) protein variants (G0, G1, and G2) in blood plasma for identification of high and low renal risk genotypes. Following trypsin digestion of liquid or dry plasma, surrogate peptides specific to each ApoL1 variant were detected by liquid chromatography-tandem mass spectrometry along with two surrogate peptides common among all variants which served as internal (positive) controls to verify correct sample processing. Using isotopically labeled peptide internal standards, the presence or absence of each surrogate peptide was determined using multiple objective metrics including: 1) retention time confirmation relative to its internal standard, 2) comparison of the internal standard-normalized response relative to pre-established thresholds for confident detection, and 3) ion ratio monitoring. Based on the pattern of variant-specific surrogate peptides detected, the genotype was accurately inferred. The final, optimized method was fully validated for liquid plasma specimens, as well as dry plasma specimens collected on a laminar flow blood separation device. For both specimen types, the latter which can be self-collected for remote or in-home sampling, the assay was shown to be reproducible, interference-free with the exception of gross hemolysis, and accurate relative to Sanger sequencing (100% agreement). This targeted, qualitative bottom-up proteomic assay for detection of ApoL1 variants in blood provides a high-throughput, cost-effective alternative to molecular methods and has potential implications to improve organ allocation by facilitating screening kidney donors for high-risk ApoL1 genotypes, but could be applicable for genotyping other clinically relevant blood proteins variants.

中文翻译：

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质量重于数量：载脂蛋白 L1 基因分型的定性、有针对性的自下而上蛋白质组学方法

开发了一种有针对性的自下而上的蛋白质组学测定，用于定性检测血浆中的载脂蛋白 L1 (ApoL1) 蛋白质变异体（G0、G1 和 G2），以识别高和低肾脏风险基因型。胰蛋白酶消化液体或干血浆后，通过液相色谱-串联质谱法检测每种 ApoL1 变体特异的替代肽以及所有变体中常见的两种替代肽，作为内部（阳性）对照，以验证样品处理是否正确。使用同位素标记的肽内标，使用多个客观指标确定每个替代肽的存在或不存在，包括：1) 相对于其内标的保留时间确认，2) 内标归一化响应相对于预先设定的阈值的比较用于可靠检测，以及 3) 离子比率监测。根据检测到的变体特异性替代肽的模式，可以准确推断基因型。最终的优化方法针对液体血浆样本以及层流血液分离装置上收集的干血浆样本进行了充分验证。对于这两种样本类型（后者可以自行采集用于远程或家庭采样），该检测结果显示具有可重复性、除严重溶血外无干扰，并且相对于桑格测序准确（100% 一致） 。这种用于检测血液中 ApoL1 变异的有针对性、定性的自下而上的蛋白质组学检测为分子方法提供了一种高通量、经济高效的替代方案，并且通过促进筛选肾脏捐献者的高风险 ApoL1 基因型，对改善器官分配具有潜在影响，但可适用于其他临床相关血液蛋白变体的基因分型。