In-depth coverage of proteomic analysis could enhance our understanding to the mechanism of the protein functions. Unfortunately, many highly hydrophobic and low-abundance proteins are easily lost during sample preparation, mainly attributed to the fact that very few extractants can simultaneously satisfy the requirements on strong solubilizing ability to membrane proteins and enzyme compatibility. Thus, it is urgent to screen ideal extractants from the huge compound libraries. Herein, molecular dynamics simulation was established to elucidate the underlying effects of different extractants on the solubilization of membrane proteins and the maintenance of trypsin activity. Taking this tool, the interaction mechanism between ionic liquids and proteins was elucidated for the first time. And 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) was found to be the suitable candidate, in accordance with our previous empirical result. Furthermore, inspired by the advantages of C12Im-Cl, an ionic liquid-based filter-aided sample preparation (i-FASP) method was developed. Using this strategy, over 3,300 proteins were confidently identified from 103 HeLa cells (~100 ng proteins) in a single run, an improvement of 53% over the conventional FASP method. Then the i-FASP method was further successfully applied to the label-free relative quantitation of human liver cancer and para-carcinoma tissues with obviously improved accuracy, reproducibility and coverage than the commonly used urea-based FASP method. All above results demonstrate i-FASP is a versatile tool for the in-depth coverage proteomic analysis of biological samples.
[doi:10.25345/C5BP75]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: ionic liquid ; deep coverage proteome analysis
Principal Investigators: (in alphabetical order) |
Lihua Zhang, Dalian Institute of Chemical Physics, China |
Submitting User: | feifang |
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