Variance Component Analysis of a Multi-Site Study for the Reproducibility of Multiple Reaction Monitoring Measurements of Peptides in Human Plasma

Background: In the Addona et al. paper (Nature Biotechnology 2009), a large-scale multi-site study was performed to quantify Multiple Reaction Monitoring (MRM) measurements of proteins spiked in human plasma. The unlabeled signature peptides derived from the seven target proteins were measured at nine different concentration levels, and their isotopic counterparts were served as the internal standards. Methodology/Principal Findings: In this paper, the sources of variation are analyzed by decomposing the variance into parts attributable to specific experimental factors: technical replicates, sites, peptides, transitions within each peptide, and higher-order interaction terms based on carefully built mixed effects models. The factors of peptides and transitions are shown to be major contributors to the variance of the measurements considering heavy (isotopic) peptides alone. For the light (12C) peptides alone, in addition to these factors, the factor of study*peptide also contributes significantly to the variance of the measurements. Heterogeneous peptide component models as well as influence analysis identify the outlier peptides in the study, which are then excluded from the analysis. Using a log-log scale transformation and subtracting the heavy/isotopic peptide [internal standard] measurement from the peptide measurements (i.e., taking the logarithm of the peak area ratio in the original scale establishes that), the MRM measurements are overall consistent across laboratories following the same standard operating procedures, and the variance components related to sites, transitions and higher-order interaction terms involving sites have greatly reduced impact. Thus the heavy peptides have been effective in reducing apparent inter-site variability. In addition, the estimates of intercepts and slopes of the calibration curves are calculated for the sub-studies. Conclusions/Significance: The MRM measurements are overall consistent across laboratories following the same standard operating procedures, and heavy peptides can be used as an effective internal standard for reducing apparent inter-site variability. Mixed effects modeling is a valuable tool in mass spectrometry-based proteomics research.