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

• DOI: 10.1038/s41467-019-13973-x
• Publication Date: 2020-01-09T11:02:26Z
• Authors: 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
• Keywords: Proteomics; 0303 health sciences; Proteome; Science; Q; 610 Medicine & health; 1600 General Chemistry; 3100 General Physics and Astronomy; Article; ddc:; 03 medical and health sciences; 1300 General Biochemistry, Genetics and Molecular Biology; Tandem Mass Spectrometry; Cell Line, Tumor; Humans; 10046 Balgrist University Hospital, Swiss Spinal Cord Injury Center; Amino Acid Sequence; Peptides; Chromatography, Liquid; HeLa Cells

AbstractNano-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.