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.