Journal Article

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

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

2020

Overview

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. Mass spectrometry-based proteomics typically relies on highly sensitive nano-flow liquid chromatography (LC) but this can reduce robustness and reproducibility. Here, the authors show that micro-flow LC enables robust and reproducible high-throughput proteomics experiments at a very moderate loss of sensitivity.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 631/1647/2067 / 631/1647/2196/1380 / 631/1647/296 / 631/45/475 / 82/16 / 82/58 / Amino Acid Sequence / Body fluids / Cell Line, Tumor / Cell lines / Chromatography / Chromatography, Liquid - methods / Coefficient of variation / Drug interaction / Drug interactions / HeLa Cells / Humanities and Social Sciences / Humans / Liquid chromatography / Mass spectrometry / Mass spectroscopy / multidisciplinary / Multiplexing / Peptides / Peptides - analysis / Proteins / Proteome - analysis / Proteomes / Proteomics / Proteomics - methods / Quantitative analysis / Reproducibility / Retention time / Robustness / Science / Science (multidisciplinary) / Scientific imaging / Sensitivity / Spectroscopy / Tandem Mass Spectrometry - methods