Quality over Quantity: A Qualitative, Targeted Bottom-up Proteomics Approach to Genotyping Apolipoprotein L1.

Quality over Quantity: A Qualitative, Targeted Bottom-up Proteomics Approach to Genotyping Apolipoprotein L1. Clin Biochem. 2020 Mar 28;: Authors: Norris Bradley M, Shufor CM, Holland PL, Levandoski M, Grant RP Abstract 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-coll...
Source: Clinical Biochemistry - Category: Biochemistry Authors: Tags: Clin Biochem Source Type: research