title: Comparison of sodium dodecyl sulfate depletion techniques for proteome analysis by mass spectrometry.
abstract: In proteomics, sodium dodecyl sulfate (SDS) is favored for protein solubilization and mass-based separation (e.g. GELFrEE or SDS PAGE). Numerous SDS depletion techniques are available to purify proteins ahead of mass spectrometry. The effectiveness of the purification has a controlling influence on the success of the analysis. Here we quantitatively assess eight approaches to SDS depletion: in-gel digestion; protein precipitation in acetone or with TCA; detergent precipitation with KCl; strong cation exchange; protein level and peptide level purification with Pierce detergent removal cartridges; and FASP II. Considering protein purity, FASP II showed the highest degree of SDS removal, matching that of in-gel digestion (over 99.99% depleted). Other methods (acetone, strong cation exchange, Pierce cartridges) also deplete SDS to levels amenable to LC-MS (>99%). Accounting for protein recovery, FASP II revealed significant sample loss (<40% yield); other approaches show even greater protein loss. We further assessed acetone precipitation, having the highest protein recovery relative to FASP II, to process GELFrEE fractionated Escherichia coli ahead of bottom-up mass spectrometry. Acetone precipitation yielded a 17% average increase in identified proteins, and 40% increase in peptides, indicating this approach as a favored strategy for SDS depletion in a proteomics workflow.
authors.affiliation: Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Carolyn
authors.initials: C
authors.lastname: Kachuk
authors.affiliation: Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Kegan
authors.initials: K
authors.lastname: Stephen
authors.affiliation: Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada. Electronic address: alan.doucette@dal.ca.
authors.forename: Alan
authors.initials: A
authors.lastname: Doucette
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D003902:Detergents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*
chemical_list: D003902:Detergents; D010455:Peptides; D011506:Proteins; D020543:Proteome; D012967:Sodium Dodecyl Sulfate
keywords: Acetone precipitation; FASP II; Protein purification; Protein recovery; Proteomics; SDS depletion