Interlaboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma

Abstract

The inability to quantify large numbers of proteins in tissues and biofluids with high precision, sensitivity, and throughput is a major bottleneck in biomarker studies. We previously demonstrated that coupling immunoaffinity enrichment using anti-peptide antibodies (SISCAPA) to multiple reaction monitoring mass spectrometry (MRM-MS) produces Immunoprecipitation MRM-MS (immuno-MRM-MS) assays that can be multiplexed to quantify proteins in plasma with high sensitivity, specificity, and precision. Here we report the first systematic evaluation of the interlaboratory performance of multiplexed (8-plex) immuno-MRM-MS in three independent labs. A staged study was carried out in which the effect of each processing and analysis step on assay coefficient of variance, limit of detection, limit of quantification, and recovery was evaluated. Limits of detection were at or below 1 ng/ml for the assayed proteins in 30 l of plasma. Assay reproducibility was acceptable for verification studies, with median intra-and interlaboratory coefficients of variance above the limit of quantification of 11% and <14%, respectively, for the entire immuno-MRM-MS assay process, including enzymatic digestion of plasma. Trypsin digestion and its requisite sample handling contributed the most to assay variability and reduced the recovery of target peptides from digested proteins. Using a stable isotope-labeled protein as an internal standard instead of stable isotope-labeled peptides to account for losses in the digestion process nearly doubled assay accuracy for this while improving assay precision 5%. Our results demonstrate that multiplexed immuno-

Interlaboratory Agreement of Insulin-like Growth Factor 1 Concentrations Measured by Mass Spectrometry

“…Variation from the sample preparation steps was minimized through the use of [ 15 N]-IGF-1 protein, which has been demonstrated previously to improve interlaboratory agreement (33 ). The use of different instrumentation contributed to a low level of variability, while reconstitution of the reference material and method of calibration were identified as the primary source of interlaboratory variation.…”

“…For example, when unfractionated plasma was digested with trypsin and supplemented with predigested, purified proteins and stable isotope-labeled peptide internal standards, the interlaboratory imprecision was Ͻ32% (31,32 ). More recently, interlaboratory imprecision was evaluated for the SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies) method of protein quantification (33 ). When unfractionated plasma was supplemented with protein standards, digested, and quantified by use of the SISCAPA method, the imprecision was Ͻ14%.…”

Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics

“…Furthermore, currently available approaches typically rely on the use of a single reference peptide or tag that is spiked early in the analytical process. Although such a reference tag corrects for the concomitant loss in endogenous peptides during sample digestion and fractionation and thus increases accuracy and reproducibility 20,21 , the use of only one reference tag requires that the quantification of each protein standard be accomplished separately. This ultimately adds avoidable steps into the workflow, potentially introducing undesirable artifacts (Supplementary Table 1).…”

Applications of targeted proteomics in systems biology and translational medicine

“…Targeted proteomics is best suited to meet these needs. Multiple studies clearly demonstrated the reproducibility of SRM‐MS across laboratories 29, 30. SRM‐MS has been applied to quantify protein levels of liver tissue across 40 strains of BDX mouse 31 and quantitative trait analysis (QTL) of 78 Saccharomyces cerevisiae strains 32.…”