An Optimized Shotgun Strategy for the Rapid Generation of Comprehensive Human Proteomes [Reanalysis: PXD004452]

Here, we present a readily approachable, optimized method for generating deep proteomes. This method takes full advantage of the high resolution that offline HpH fractionation provides and combines it with short gradients and a fast 20 Hz scanning tandem MS (MS/MS) method for subsequent proteome analysis. This combination solves the problems of lower sensitivity usually associated with fast scanning MS/MS methods, and we demonstrate that the concept of running many fractions on short gradients is the sweet-spot in terms of best compromise between instrument time used and sequencing depth obtained. To benchmark this workflow we analyzed the human proteome of the HeLa cervix carcinoma cell line, which is the most commonly used model for studying human cell biology (Masters, 2002).[Original project description]