In-Depth Proteome Coverage by Improving Efficiency for Membrane Proteome Analysis.

Although great achievement has been made in the mapping of human proteome, the efficiency of sample preparation still needs to be improved, especially for membrane proteins. Herein, we presented a novel method to deepen proteome coverage by the sequential extraction of proteins using urea and 1-dodecyl-3- methylimidazolium chloride (C12Im-Cl). With such a strategy, the commonly lost hydrophobic proteins by 8 M urea extraction could be further recovered by C12Im-Cl, as well as the suppression effect of high abundance soluble proteins could be decreased. Followed by the in situ sample preparation and separation with different stationary phases, more than 9810 gene products could be identified, covering 8 orders of magnitude in abundance, which was, to the best of our knowledge, the largest data set of HeLa cell proteome. Compared with previous work, not only the number of proteins identified was obviously increased, but also the analysis time was shortened to a few days. Therefore, we expect that such a strategy has great potential applications to achieve unprecedented coverage for proteome analysis.