Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC-MS/MS

Nat Commun. 2020 Jan 9;11(1):157. doi: 10.1038/s41467-019-13973-x.


Nano-flow liquid chromatography tandem mass spectrometry (nano-flow LC-MS/MS) is the mainstay in proteome research because of its excellent sensitivity but often comes at the expense of robustness. Here we show that micro-flow LC-MS/MS using a 1 × 150 mm column shows excellent reproducibility of chromatographic retention time (<0.3% coefficient of variation, CV) and protein quantification (<7.5% CV) using data from >2000 samples of human cell lines, tissues and body fluids. Deep proteome analysis identifies >9000 proteins and >120,000 peptides in 16 h and sample multiplexing using tandem mass tags increases throughput to 11 proteomes in 16 h. The system identifies >30,000 phosphopeptides in 12 h and protein-protein or protein-drug interaction experiments can be analyzed in 20 min per sample. We show that the same column can be used to analyze >7500 samples without apparent loss of performance. This study demonstrates that micro-flow LC-MS/MS is suitable for a broad range of proteomic applications.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Line, Tumor
  • Chromatography, Liquid / methods*
  • HeLa Cells
  • Humans
  • Peptides / analysis
  • Proteome / analysis*
  • Proteomics / methods*
  • Tandem Mass Spectrometry / methods*


  • Peptides
  • Proteome