A theoretical foundation of the target-decoy search strategy for false discovery rate control in proteomics

TL;DR

This paper provides a rigorous statistical analysis of the target-decoy search strategy in proteomics, proving its conservative nature and proposing modifications for accurate FDR control at the peptide level.

Contribution

It establishes the theoretical foundation of TDS, demonstrating its limitations and proposing a modified FDR estimation method for peptide-level control.

Findings

Concatenated TDS provides a conservative FDR estimate.

Modified FDR formula enables rigorous peptide-level FDR control.

Spectrum-level FDR control remains challenging.

Abstract

Motivation: Target-decoy search (TDS) is currently the most popular strategy for estimating and controlling the false discovery rate (FDR) of peptide identifications in mass spectrometry-based shotgun proteomics. While this strategy is very useful in practice and has been intensively studied empirically, its theoretical foundation has not yet been well established. Result: In this work, we systematically analyze the TDS strategy in a rigorous statistical sense. We prove that the commonly used concatenated TDS provides a conservative estimate of the FDR for any given score threshold, but it cannot rigorously control the FDR. We prove that with a slight modification to the commonly used formula for FDR estimation, the peptide-level FDR can be rigorously controlled based on the concatenated TDS. We show that the spectrum-level FDR control is difficult. We verify the theoretical conclusions with real mass spectrometry data.