Top-Down and Bottom-Up Proteomics of SDS-Containing Solutions Following Mass-Based Separation

SDS has recognized benefits for protein sample preparation, including solubilization and imparting molecular weight separation (e.g., SDS-PAGE). Here, we compare two proteome workflows which incorporate SDS for protein separation, namely, SDS-PAGE coupled to LC/MS (GeLC MS), along with a solution separation platform, GELFrEE, for intact proteome prefractionation and identification. Despite the clear importance of SDS in these and other proteome analysis workflows, the affect of SDS on an LC/MS proteome experiment has not been quantified. We first examined the influence of SDS on both a bottom-up as well as a top-down (intact protein) MS workflow. Surprisingly, at levels up to 0.01% SDS in the injected sample, reliable MS characterization is obtained. We subsequently explored organic precipitation protocols (chloroform/methanol/water and acetone) as a means of lowering SDS, and present a simple modified acetone precipitation protocol which consistently enables MS proteome characterizations from samples ini...