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


Yangyang Bian, Runsheng Zheng, Florian P. Bayer, Cassandra Wong, Yun-Chien Chang, Chen Meng, Daniel P. Zolg, Maria Reinecke, Jana Zecha, Svenja Wiechmann, Stephanie Heinzlmeir, Johannes Scherr, Bernhard Hemmer, Mike Baynham, Anne-Claude Gingras, Oleksandr Boychenko, Bernhard Kuster

Nat Commun.2020, 11, 157

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 × 150mm 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.