Robust micro-flow LC-MS/MS for proteome analysis – 38,000 runs and counting

Micro-flow liquid chromatography tandem mass spectrometry (µLC–MS/MS) is increasingly becoming an alternative to nano-LC–MS/MS for the analysis of proteomes. We have recently demonstrated the potential of a µLC–MS/MS system operating with a 1 mm i.d. × 150 mm column and at a flow rate of 50 µl/min for high-throughput applications. Based on the analysis of ~38,000 samples measured on two instruments over the past two years, we show that the approach is extremely robust. Up to 1,500 analysis were performed within one month and >14,000 samples could be analyzed on a single column without loss of chromatographic performance. Samples included proteomes of cell lines, animal tissue and human body fluids, which were analyzed either with or without prior peptide fractionation or stable isotope labeling. We show that the µLC–MS/MS system is capable of measuring ~2,700 proteins from human plasma and ~5,200 proteins from human urine within one day, demonstrating its potential for in-depth as well as high-throughput clinical application.