Tandem mass spectrometry (MS/MS) combined with database search methods can identify the proteins present in complex mixtures. High-throughput methods that infer probable peptide sequences from enzymatically digested protein samples create a challenge in how best to aggregate the evidence for candidate proteins. Typically the results of multiple technical and/or biological replicate experiments must be combined in order to maximize sensitivity. We present a statistical method for estimating probabilities of protein expression that integrates peptide sequence identifications from multiple search algorithms and replicate experimental runs. The method was applied to create a repository of 797 non-homologous zebrafish (Danio rerio) proteins, at an empirically-validated false identification rate under 1%, as a resource for the development of targeted quantitative proteomics assays. We have implemented this statistical method as an analytic module that can be integrated with an existing suite of open-source proteomics software.