In the last decade, a revolution in liquid chromatography-mass spectrometry (LC-MS) based proteomics was unfolded with the introduction of dozens of novel instruments that incorporate additional data dimensions through innovative acquisition methodologies, in turn inspiring specialized data analysis pipelines. Simultaneously, a growing number of proteomics datasets have been made publicly available through data repositories such as ProteomeXchange, Zenodo and Skyline Panorama. However, developing algorithms to mine this data and assessing the performance on different platforms is currently hampered by the lack of a single benchmark experimental design. Therefore, we acquired a hybrid proteome mixture on different instrument platforms and in all currently available families of data acquisition. Here, we present a comprehensive Data-Dependent and Data-Independent Acquisition (DDA/DIA) dataset acquired using several of the most commonly used current day instrumental platforms. The dataset consists of over 700 LC-MS runs, including adequate replicates allowing robust statistics and covering over nearly 10 different data formats, including scanning quadrupole and ion mobility enabled acquisitions. Datasets are available via ProteomeXchange (PXD028735).

Here, we created a comprehensive dataset on a single benchmark experimental design using samples composed of commercial Human, Yeast and E.coli full proteome digests, adapted from Navarro et al. It contains a ground truth that serves as a quality control for bioinformatics algorithm validation. This sample was acquired in adequate replicates on many of the current day instrumental platforms – partially in nano flow LC and partially in capillary flow LC - by most of the available acquisition strategy families, covering all commonly measured ion coordinates

Two hybrid proteome samples A and B containing known quantities of Human, Yeast and E.coli tryptic peptides, as described by Navarro et al. were prepared in three consecutive times to include handling variability. Additionally, a QC sample was created by mixing one sixth of each of the six master batches (65% w/w Human, 22.5% w/w Yeast and 12.5% w/w E.coli). These commercial lysates were measured individually and as triple hybrid proteome mixtures each in triplicate using DDA and DIA acquisition methodologies available on six LC-MS/MS platforms, i.e. SCIEX TripleTOF5600 and TripleTOF 6600+, Thermo Orbitrap QE HF-X, Waters Synapt G2-Si and Synapt XS and Bruker timsTOF Pro.