A robotic protocol for high-throughput processing of samples for selected reaction monitoring assays.

Selected reaction monitoring mass spectrometry (SRM-MS) is a sensitive and accurate method for the quantification of targeted proteins in biological specimens. However, the sample throughput and reliability of this technique is limited by the complexity of sample preparation, as well as instrumentation and data processing. Modern robotic equipment allows for rapid and accurate processing of large number of samples and makes SRM-MS assay applicable in epidemiological studies. Herein, we describe an automated sample processing platform developed in the context of an SRM-MS protocol for the assay of complement factor H protein and its variants in human plasma. We report detailed performance data on plasma digestion, sample cleanup and optimized robotic handling implemented on a Biomek® NXp Workstation. Method validation was assessed with isotopically labeled peptide standards and had high reproducibility of intra-day assay (CVs from 2.7 to 17.5% with median CV of 5.3%) and inter-day assay (CVs from 4.8 to 17.6 with median CV of 7.2%) for all peptides.

A targeted proteomic assay for the measurement of plasma proteoforms related to human aging phenotypes.

Circulating polypeptides and proteins have been implicated in reversing or accelerating aging phenotypes, including growth/differentiation factor 8 (GDF8), GDF11, eotaxin, and oxytocin. These proteoforms, which are defined as the protein products arising from a single gene due to alternative splicing and PTMs, have been challenging to study. Both GDF8 and GDF11 have known antagonists such as follistatin (FST), and WAP, Kazal, immunoglobulin, Kunitz, and NTR domain-containing proteins 1 and 2 (WFIKKN1, WFIKKN2). We developed a novel multiplexed SRM assay using LC-MS/MS to measure five proteins related to GDF8 and GDF11 signaling, and in addition, eotaxin, and oxytocin. Eighteen peptides consisting of 54 transitions were monitored and validated in pooled human plasma. In 24 adults, the mean (SD) concentrations (ng/mL) were as follows: GDF8 propeptide, 11.0 (2.4); GDF8 mature protein, 25.7 (8.0); GDF11 propeptide, 21.3 (10.9); GDF11 mature protein, 16.5 (12.4); FST, 29.8 (7.1); FST cleavage form FST303, 96.4 (69.2); WFIKKN1, 38.3 (8.3); WFIKKN2, 32.2 (10.5); oxytocin, 1.9 (0.9); and eotaxin, 2.3 (0.5). This novel multiplexed SRM assay should facilitate the study of the relationships of these proteoforms with major aging phenotypes.

A novel, multiplexed targeted mass spectrometry assay for quantification of complement factor H (CFH) variants and CFH-related proteins 1-5 in human plasma.

Age-related macular degeneration (AMD) is a leading cause of visual loss among older adults. Two variants in the complement factor H (CFH) gene, Y402H and I62V, are strongly associated with risk of AMD. CFH is encoded in regulator of complement activation gene cluster in chromosome 1q32, which includes complement factor related (CFHR) proteins, CFHR1 to CFHR5, with high amino acid sequence homology to CFH. Our goal was to build a SRM assay to measure plasma concentrations of CFH variants Y402, H402, I62, and V62, and CFHR1-5. The final assay consisted of 24 peptides and 72 interference-free SRM transition ion pairs. Most peptides showed good linearity over 0.3-200 fmol/µL concentration range. Plasma concentrations of CFH variants and CFHR1-5 were measured using the SRM assay in 344 adults. Plasma CFH concentrations (mean, SE in µg/mL) by inferred genotype were: YY402, II62 (170.1, 31.4), YY402, VV62 (188.8, 38.5), HH402, VV62 (144.0, 37.0), HY402, VV62 (164.2, 42.3), YY402, IV62 (194.8, 36.8), HY402, IV62 (181.3, 44.7). Mean (SE) plasma concentrations of CFHR1-5 were 1.63 (0.04), 3.64 (1.20), 0.020 (0.001), 2.42 (0.18), and 5.49 (1.55) µg/mL, respectively. This SRM assay should facilitate the study of the role of systemic complement and risk of AMD.