Data-independent Acquisition-based Proteome and Phosphoproteome Profiling across Six Melanoma Cell Lines

The rapid documentation of the steady-state gene expression landscape of the cells used in particular experiments may help to improve the reproducibility of scientific research. Here we applied a data-independent acquisition mass spectrometry (DIA-MS) method, coupled with a peptide spectral-library free data analysis workflow, to measure both proteome and phosphoproteome of a melanoma cell line panel with different metastatic properties. For each cell line, the single-shot DIA-MS detected 8,100 proteins and almost 40,000 phosphopeptides in the respective measurement of two hours. Benchmarking the DIA-MS data towards the RNA-seq data and tandem mass tag (TMT)-MS results from the same set of cell lines demonstrated comparable qualitative coverage and quantitative reproducibility. Our data confirmed the high but complex mRNA~protein and protein~phospsite correlations. The results successfully established DIA-MS as a strong and competitive proteotyping approach for cell lines.