Mix24X, a Lab-Assembled Reference to Evaluate Interpretation Procedures for Tandem Mass Spectrometry Proteotyping of Complex Samples

Correct identification of the microorganisms present in a complex sample is a crucial issue. Proteotyping based on tandem mass spectrometry can help establish an inventory of organisms present in a sample. Evaluation of bioinformatics strategies and tools for mining the recorded datasets is essential to establish confidence in the results obtained and to improve these pipelines in terms of sensitivity and accuracy. Here, we propose several tandem mass spectrometry datasets recorded on an artificial reference consortium comprising 24 bacterial species. This assemblage of environmental and pathogenic bacteria covers 20 different genera and 5 bacterial phyla. The dataset comprises difficult cases, such as the Shigella flexneri species, which is closely related to Escherichia coli, and several highly sequenced clades. Different acquisition strategies simulate real-life scenarios: from rapid survey sampling to exhaustive analysis. We provide access to individual proteomes of each bacterium separately to provide a rational basis for evaluating the assignment strategy of MS/MS spectra when recorded from complex mixtures. This resource should provide an interesting common reference for developers who wish to compare their proteotyping tools and for those interested in evaluating protein assignment when dealing with complex samples, such as microbiomes.