The bottom-up strategy for proteome analysis typically employs a multistep sample preparation workflow that suffers from being time-consuming and sample loss or contamination caused by the off-line manual operation. Herein, we developed a hollow fibre membrane (HFM)-aided fully automated sample treatment (FAST) method. Due to the confinement effects of HFMs and the immobilized enzymatic reactor, the proteome samples could be denatured, reduced, desalted and digested within 8–20 min via the one-stop service. This method also showed superiority in trace sample analysis. In one and half hours, we could identify about 1,600 protein groups for 500 HeLa cells as the starting materials, 1.5–8 times more than those obtained by previously reported methods. Through the on-line combination of FAST with nano-liquid chromatography-electrospray ionization tandem mass spectrometry (nanoLC-ESI-MS/MS), we further established a fully integrated platform for label-free quantification of proteome with high reproducibility and precision. Collectively, FAST presented here represents a major advance in the high throughput sample treatment and quantitative analysis of proteomes.
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