Data-independent acquisition-based proteome and phosphoproteome profiling across six melanoma cell lines reveals determinants of proteotypes

Mol Omics. 2021 Jun 14;17(3):413-425. doi: 10.1039/d0mo00188k.


Human cancer cell lines are widely used in pharmacological and systems biological studies. The rapid documentation of the steady-state gene expression landscape of the cells used in a particular experiment 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 the proteome and phosphoproteome of a melanoma cell line panel with different metastatic properties. For each cell line, the single-shot DIA-MS detected 8100 proteins and almost 40 000 phosphopeptides in the respective measurements 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. The data further showed that all subunits of the glycosylphosphatidylinositol (GPI)-anchor transamidase complex were overexpressed in metastatic melanoma cells and identified altered phosphoprotein modules such as the BAF complex and mRNA splicing between metastatic and primary cells. This study provides a high-quality resource for calibrating DIA-MS performance, benchmarking DIA bioinformatic algorithms, and exploring the metastatic proteotypes in melanoma cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Chromatography, Liquid
  • Computational Biology / methods*
  • Gene Expression Profiling
  • Humans
  • Melanoma / genetics
  • Melanoma / metabolism*
  • Neoplasm Metastasis
  • Phosphoproteins / analysis*
  • Phosphoproteins / genetics
  • Protein Interaction Maps*
  • Proteomics / methods*
  • Sequence Analysis, RNA
  • Tandem Mass Spectrometry


  • Phosphoproteins