Bayesian Confidence Intervals for Multiplexed Proteomics Integrate lon-Statistics with Peptide Quantification Concordance

Multiplexed proteomics has emerged as a powerful tool to measure relative protein expression levels across multiple conditions. The relative protein abundances are inferred by comparing the signal generated by isobaric tags, which encode the samples’ origins. Intuitively, the trust associated with a protein measurement depends on the similarity of ratios from the protein’s peptides and the signal level of these measurements. However, typically only the most likely results are reported without providing confidence for these measurements. Here we present a mathematically rigorous approach that integrates peptide MS-signal and peptide-measurement agreement into an estimation of the true protein ratio and the associated confidence (BACIQ). The main advantages of BACIQ are: 1) it removes the need to threshold reported peptide signal based on an arbitrary cut-off, thereby reporting more measurements from a given experiment; 2) confidence can be assigned without replicates; 3) for repeated experiments BACIQ provides confidence intervals for the union, not the intersection, of quantified proteins; 4) for repeated experiments, BACIQ confidence intervals are more predictive than confidence intervals based on protein measurement agreement. To demonstrate the power of BACIQ we reanalyzed previously published data on subcellular protein movement upon treatment with an Exportin 1 inhibiting drug. We detect ~2x more highly significant movers, down to subcellular localization changes of ~1%. Thus, our method drastically increases the value obtainable from quantitative proteomics experiments helping researchers to interpret their data and prioritize resources. To make our approach easily accessible we distribute it via a Python/Stan package. Abbreviations : RNC : Relative Nuclear Concentration FDR : False Discovery Rate TMT : Tandem Mass Tag TMT-MS3 : MultiNotch MS3 analysis of TMT samples TMTc+ : Complement Reporter Ion Quantification Method ANOVA : Analysis of variance BACIQ : Bayesian Approach to Confidence Intervals for protein Quantitation NEM : N-Ethylmaleimide BCA : bicinchoninic acid Lys-C : Lysil Endopeptidase EPPS : 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid RCF : Relative Centrifugational Force LMB : Leptomycin B Exp1 : Exportin 1