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 initially containing 2% SDS. With this effective strategy for SDS reduction, the GELFrEE MS workflow for bottom-up proteome analysis was characterized relative to GeLC MS. Remarkable agreement in the number and type of identified proteins was obtained from these two separation platforms, validating the use of SDS in solution-phase proteome analysis.
Botelho
Diane
D
Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada.
Wall
Mark J
MJ
Vieira
Douglas B
DB
Fitzsimmons
Shayla
S
Liu
Fang
F
Doucette
Alan
A
D011232:Chemical Precipitation; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D020543:Proteome / Q000032:analysis / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*
D011506:Proteins; D020543:Proteome; D012967:Sodium Dodecyl Sulfate