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

TLDR

Nano‑flow LC–MS/MS is the standard for proteomics due to its high sensitivity, though it often sacrifices robustness. The 1 × 150 mm micro‑flow LC–MS/MS system identifies >9 000 proteins and >120 000 peptides in 16 h, detects >30 000 phosphopeptides in 12 h, and analyzes protein‑protein or protein‑drug interactions in 20 min per sample, with tandem‑mass‑tag multiplexing raising throughput to 11 proteomes per 16 h. Micro‑flow LC–MS/MS delivers <0.3 % retention‑time CV and <7.5 % protein‑quantification CV across >2000 samples, supports analysis of >7500 samples without performance loss, and is suitable for diverse proteomic applications.

Abstract

Abstract 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 (&lt;0.3% coefficient of variation, CV) and protein quantification (&lt;7.5% CV) using data from &gt;2000 samples of human cell lines, tissues and body fluids. Deep proteome analysis identifies &gt;9000 proteins and &gt;120,000 peptides in 16 h and sample multiplexing using tandem mass tags increases throughput to 11 proteomes in 16 h. The system identifies &gt;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 &gt;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.