title: Comparison of sample preparation techniques for large-scale proteomics.
abstract: Numerous workflows exist for large-scale bottom-up proteomics, many of which achieve exceptional proteome depth. Herein, we evaluated the performance of several commonly used sample preparation techniques for proteomic characterization of HeLa lysates [unfractionated in-solution digests, SDS-PAGE coupled with in-gel digestion, gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) technology, SCX StageTips and high-/low-pH reversed phase fractionation (HpH)]. HpH fractionation was found to be superior in terms of proteome depth (>8400 proteins detected) and fractionation efficiency compared to other techniques. SCX StageTip fractionation required minimal sample handling and was also a substantial improvement over SDS-PAGE separation and GELFrEE technology. Sequence coverage of the HeLa proteome increased to 38% when combining all workflows, however, total proteins detected improved only slightly to 8710. In summary, HpH fractionation and SCX StageTips are robust techniques and highly suited for complex proteome analysis.
authors.affiliation: Department of Biochemistry, University of Western Ontario, London, ON, Canada.
authors.forename: Miljan
authors.initials: M
authors.lastname: Kuljanin
authors.affiliation: Department of Anatomy and Cell Biology, University of Western Ontario, London, ON, Canada.
authors.forename: Dylan Z
authors.initials: DZ
authors.lastname: Dieters-Castator
authors.affiliation: Department of Physiology and Pharmacology, University of Western Ontario, London, ON, Canada.
authors.forename: David A
authors.initials: DA
authors.lastname: Hess
authors.affiliation: Department of Anatomy and Cell Biology, University of Western Ontario, London, ON, Canada.|Department of Oncology, University of Alberta, Edmonton, AB, Canada.
authors.forename: Lynne-Marie
authors.initials: LM
authors.lastname: Postovit
authors.affiliation: Department of Biochemistry, University of Western Ontario, London, ON, Canada.
authors.forename: Gilles A
authors.initials: GA
authors.lastname: Lajoie
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*
chemical_list: D020543:Proteome
keywords: Bottom-up proteomics; Gel-eluted liquid fractionation entrapment electrophoresis (GELFrEE); High pH; SCX StageTip; SDS-PAGE; Technology