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.

尽管人类蛋白质组（proteome）图谱绘制已取得显著进展，但样品制备效率仍有待提升，针对膜蛋白的相关工作尤为如此。在此，我们提出一种全新方法，通过使用尿素与1-十二烷基-3-甲基咪唑氯盐（C12Im-Cl）进行蛋白质的连续萃取，以提升蛋白质组覆盖深度。借助该策略，经8 M尿素萃取时通常会丢失的疏水蛋白质，可通过C12Im-Cl进一步回收，同时还能削弱高丰度可溶性蛋白的抑制效应。随后结合原位样品制备与不同固定相分离技术，共可鉴定出9810余种基因产物，其丰度覆盖8个数量级；据我们所知，这是目前已报道的最大规模HeLa细胞蛋白质组数据集。与既往研究相比，本方法不仅显著提升了鉴定得到的蛋白质数量，还将分析周期缩短至数天。因此，我们预计该策略在实现蛋白质组分析的空前覆盖度方面具有巨大应用潜力。