Fig. 1

Study design and implementation. a A set of 30 SIS peptides partitioned into five groups (A–E, six peptides in each) were diluted into a HEK293 cell lysate to span a large dynamic range. Starting at a different upper concentration for each group, they were threefold diluted into the matrix to cover a concentration range from 12 amol to 10 pmol in 1 µg of cell lysate. This created a set of five samples to be run by SWATH-MS on the TripleTOF 5600/5600+ system at each site. Each sample was run once per day on day 1, 3, and 5, with the exception of sample 4 which was run 3× on each day. b After data acquisition, the 229 SWATH-MS files were assembled centrally and processed using two strategies. The SIS peptide concentration curves were assessed using MultiQuant Software, allowing for the determination of linear dynamic range (LDR), and LLOQs for each peptide. In addition, the intra- and inter-day CVs were determined before and after normalization. The HEK293 proteome matrix data was analyzed using the OpenSWATH pipeline and the Combined Human Assay Library consisting of ~10,000 proteins. The false discovery rate was controlled at the peptide query and protein level using PyProphet. Protein abundances were inferred by summing the top five most abundant fragment ions from the three most abundant peak groups using the aLFQ software. We then used protein abundances to cluster, and compute Pearson correlation coefficients, for all samples from all sites