Sample preparation is a critical step to ensure the sensitivity,accuracy,precision and throughput of both qualitative and quantitative proteome analysis.Bottom-up proteomics typically employs multistep sample preparation workflow,which is subjected to long-time manual operation,sample loss and contamination.To solve these problems,we developed a hollow fiber membrane-aided fully automated sample treatment(FAST)method,by which proteomic samples could be denatured,reduced,desalted and digested within 6-20 min via "one-stop" service.Through the on-line combination of FAST with nano-liquid chromatography-electrospray ionization tandem mass spectrometry(nLC-ESI-MS/MS),we further established a fully integrated platform for high-throughput proteome quantification.Furthermore,efficient extraction and processing of trace proteins from complex matrices is also a scientific issue of widespread concern in proteomics research.To meet these requirements,we developed a new proteomic sample preparation method,called as solid-phase alkylation(SPA),in which detergent-solubilized proteins were first reduced by tris(2-carboxyethyl)phosphine hydrochloride(TCEP),and then covalently bound onto the idoacetic acid brushes grafted on silica microspheres as protein extraction sorbents and micro-reactor,finally the detergent and other interferences in proteins were completely removed by extensive washing of proteins conjugated on the solid supports with methanol and buffer solution,followed by highly efficient on-beads protein digestion.Compared to conventional liquid-phase based sample preparation protocols,the solid-phase method did not add any additional sample preparation steps,and exhibited many advantages,allowing single-run analyses of trace samples with high throughput and deep proteome coverage.