Comparison between procedures using SDS for shotgun proteomic analyses of complex samples

• Published in: Proteomics (Weinheim) Vol. 11; no. 14; pp. 2931 - 2935
• Main Authors: Bereman, Michael S., Egertson, Jarrett D., MacCoss, Michael J.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01.07.2011; WILEY‐VCH Verlag; Wiley-VCH; Wiley Subscription Services, Inc
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Bottom-up proteomics; Caenorhabditis elegans; Caenorhabditis elegans Proteins - analysis; Fundamental and applied biological sciences. Psychology; HEK293 Cells - chemistry; Humans; Mass Spectrometry - methods; Miscellaneous; Peptides; Peptides - analysis; Protein identifications; Proteins; Proteins - analysis; Proteomics - methods; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins - analysis; Sample preparation protocols; Shotgun proteomics; Sodium dodecyl sulfate; Sodium Dodecyl Sulfate - chemistry; Spectrum analysis; Surface-Active Agents - chemistry; Technology; Sodium dodecyl sulfate; Sample preparation; Sodium; Technology; Proteomics; Identification; Shotgun proteomics; Protein; Lauryl sulfate; Bottom-up proteomics; Protein identifications; Sample preparation protocols
• ISSN: 1615-9853; 1615-9861; 1615-9861
• DOI: 10.1002/pmic.201100045

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.