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A Surfactant Cocktail-Aided Extraction/Precipitation/On-Pellet Digestion (SEPOD) Strategy Enables Rapid, Efficient and Reproducible Sample Preparation for Large-Scale Quantitative Proteomics Shichen Shen, Bo An, Xue Wang, Shannon P. Hilchey, Jun Li, Jin Cao, Yu Tian, Chenqi Hu, Liang Jin, Andrew Ng, Chengjian Tu, Miao Qu, Martin ZAnd, and Jun Qu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02172 • Publication Date (Web): 05 Aug 2018 Downloaded from http://pubs.acs.org on August 6, 2018
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Analytical Chemistry
A Surfactant Cocktail-Aided Extraction/Precipitation/On-Pellet Digestion (SEPOD) Strategy Enables Rapid, Efficient and Reproducible Sample Preparation for Large-Scale Quantitative Proteomics #
Shichen Shen1,2, #Bo An1,2, Xue Wang2,3, Shannon P. Hilchey4, Jun Li1,2, Jin Cao5, Yu Tian6,
Chenqi Hu6, Liang Jin6, Andrew Ng2,7, Chengjian Tu1,2, *Miao Qu8, *Martin S. Zand4, *Jun Qu1,2 1
Department of Pharmaceutical Sciences, SUNY at Buffalo, Buffalo, NY 14214;
2
New York State
Center of Excellence in Bioinformatics & Life Sciences, Buffalo, NY, Buffalo, NY 14203; Park Cancer Institute, Buffalo, NY, Buffalo, NY 14263; Rochester Medical Center, Rochester, NY 14642;
5
4
3
Roswell
Division of Nephrology, University of
National Institute for Food and Drug Control,
Beijing, China 100050; 6AbbVie Bioresearch Center Inc., Worcester, MA 01605; 7 School of Dental Medicine, SUNY at Buffalo, Buffalo, NY 14214; 8 Department of Neurology, XuanWu Hospital, Capital University of Medicine, Beijing, China 100053;
#
The authors contributed equally to this article.
*
Corresponding Authors
Miao Qu, M.D., Ph.D. Department of Neurology, XuanWu Hospital, Capital University of Medicine, 45 Changchun Street, Beijing, China 100053 Phone: +86 13621104599 E-mail:
[email protected] ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Martin S. Zand, M.D., Ph.D. Division of Nephrology, Strong Memorial Hospital 601 Elmwood Ave, AC-3 University of Rochester Medical Center, Rochester, NY 14642 Phone: (585)273-2819 E-mail:
[email protected] Jun Qu, Ph.D. Department of Pharmaceutical Sciences, 318 Kapoor Hall, State University of New York at Buffalo, Buffalo, NY 14214-1200 Phone: (716) 645-4821 Fax: (716) 645-3693 E-mail:
[email protected] ACS Paragon Plus Environment
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Analytical Chemistry
Abstract For quantitative proteomics, efficient, robust, and reproducible sample preparation with high throughput is critical yet challenging, especially when large cohorts are involved, as is often required by clinical/pharmaceutical studies. We describe a rapid and straightforward surfactant cocktail-aided extraction/precipitation/on-pellet digestion(SEPOD) strategy to address this need. Prior to organic solvent precipitation and on-pellet digestion, SEPOD treats samples with a surfactant cocktail(SC) containing multiple non-ionic/anionic surfactants, which achieves: i)exhaustive/reproducible protein extraction, including membrane-bound proteins; ii)effective removal of detrimental non-protein matrix components(e.g.>94%
of phospholipids);
iii)rapid/efficient
proteolytic
digestion
owing
to
dual(surfactants+precipitation) denaturation. The optimal SC composition and concentrations were determined by Orthogonal-Array-Design investigation of their collective/individuals effects on protein extraction/denaturation. Key parameters for cleanup and digestion were experimentally identified as well. The optimized SEPOD procedures allowed a rapid 6h digestion providing a clean digest with high peptide yields and excellent quantitative reproducibility(esp.low-abundance proteins). Compared with filter-assisted sample preparation (FASP) and In-solution digestion, SEPOD showed superior performance by recovering substantially more peptide/proteins(including integral membrane proteins), yielding significantly higher peptide intensities, and improving quantification for peptides with extreme physicochemical properties. SEPOD was further applied in a large-cohort temporal investigation of 44 IAV-infected mouse lungs, providing efficient and reproducible peptide yields(77.9±4.6 %) across all samples. With the IonStar pipeline, >6,400 unique protein groups were quantified(≥2-peptide/protein, peptide-FDR
