title: Comparison between procedures using SDS for shotgun proteomic analyses of complex samples.
abstract: Filter-aided sample preparation (FASP) and a new sample preparation method using a modified commercial SDS removal spin column are quantitatively compared in terms of their performance for shotgun proteomic experiments in three complex proteomic samples: a Saccharomyces cerevisiae lysate (insoluble fraction), a Caenorhabditis elegans lysate (soluble fraction), and a human embryonic kidney cell line (HEK293T). The characteristics and total number of peptides and proteins identified are compared between the two procedures. The SDS spin column procedure affords a conservative fourfold improvement in throughput, is more reproducible, less expensive (i.e. requires less materials), and identifies between 30 and 107% more peptides at q≤0.01, than the FASP procedure. The peptides identified by SDS spin column are more hydrophobic than species identified by the FASP procedure as indicated by the distribution of GRAVY scores. Ultimately, these improvements correlate to as great as a 50% increase in protein identifications with two or more peptides.
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Bereman
authors.forename: Jarrett D
authors.initials: JD
authors.lastname: Egertson
authors.forename: Michael J
authors.initials: MJ
authors.lastname: MacCoss
mesh_terms: D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000032:analysis; D057809:HEK293 Cells / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D013501:Surface-Active Agents / Q000737:chemistry*
chemical_list: D029742:Caenorhabditis elegans Proteins; D010455:Peptides; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate