Mass spectrometry (MS)-based proteome profiling is essential for molecular diagnostics in modern biomedical study. To date, sample preparation including protein extraction and proteolysis is still very challenging and lack of efficiency. Recently tips-based sample preparation protocols exhibit strong potentials to achieve the goal of "a proteome in an hour". However, in-tip proteolysis is still rarely reported and far from ideal for dealing with complex bio-samples. In this work, nanoreactors encapsulated micropipette tips were demonstrated as high performance devices for fast (similar to minutes) and multiplexing proteolysis to assist the profiling of cancer cells proteome. Nanoporous silica materials with controlled pore size and surface chemistry were prepared as nanoreactors and encapsulated in micropipette tips for efficient in situ proteolysis. The as-constructed device showed desirable sensitivity (LOD of 0.204 +/- 0.008 mu g/mL and LOQ of 0.937 +/- 0.055 mu g/mL), selectivity, stability (two months under -20 degrees C), reusability (at least 10 times), and little memory effect in MS based bottom-up proteomic analysis. It was used for comprehensive protein mapping from cancer cell lines. The number of identified proteins was increased by 18%, 22%, 52%, and 52% dealing with HepG2, F56, MCF7, and HCCLM3 cancer cells, compared to traditional in-solution proteolysis based bottom-up proteomic strategy. With the enhanced performance, our work built a novel, efficient and miniaturized platform for facile proteomic sample preparation, which is promising for advanced biomarkers discovery in biomedical study. (C) 2016 Published by Elsevier B.V.
[1]Fudan Univ, Inst Biomed Sci, Dept Chem, State Key Lab Mol Engn Polymers, Shanghai 200433, Peoples R China.
[2]Shanghai Jiao Tong Univ, Med X Res Inst, Sch Biomed Engn, Shanghai 200030, Peoples R China.
(8.0+极速模式)