The Journal of Membrane Biology

, Volume 247, Issue 9–10, pp 941–947 | Cite as

APols-Aided Protein Precipitation: A Rapid Method for Concentrating Proteins for Proteomic Analysis

  • Zhibin Ning
  • Brett Hawley
  • Deeptee Seebun
  • Daniel FigeysEmail author


Amphipols (APols) are a newly designed and milder class of detergent. They have been used primarily in protein structure analysis for membrane protein trapping and stabilization. We have recently demonstrated that APols can be used as an alternative detergent for proteome extraction and digestion, to achieve a “One-stop” single-tube workflow for proteomics. In this workflow, APols are removed by precipitation after protein digestion without depleting the digested peptides. Here, we took further advantage of this precipitation characteristic of APols to concentrate proteins from diluted samples. In contrast with tryptic peptides, a decrease in pH leads to the unbiased co-precipitation of APols with proteins, including globular hydrophilic proteins. We demonstrated that this precipitation is a combined effect of acid precipitation and the APols’ protein interactions. Also, we have been able to demonstrate that APols-aided protein precipitation works well on diluted samples, such as secretome sample, and provides a rapid method for protein concentration.


Amphipols Proteomics Protein precipitation Concentrating protein Mass spectrometry 



D. F. acknowledges a Canada Research Chair in Proteomics and Systems Biology. Funding for this project was provided by NSERC-Canada. Z. N. acknowledges a postdoctoral scholarship from the CIHR Training Program in Neurodegenerative Lipidomics (TGF-96121). Z. N. would also like to acknowledge Dr. Jean-Luc POPOT for the valuable discussion and advices, and Alexandra Therese Star’s help for proofing.

Supplementary material

232_2014_9668_MOESM1_ESM.docx (957 kb)
Supplementary material 1 (DOCX 956 kb)
232_2014_9668_MOESM2_ESM.xlsx (327 kb)
Supplementary material 2 (XLSX 327 kb)
232_2014_9668_MOESM3_ESM.xlsx (693 kb)
Supplementary material 3 (XLSX 692 kb)


  1. Bechara C, Bolbach G, Bazzaco P, Sharma KS, Durand G, Popot JL, Zito F, Sagan S (2012) MALDI-TOF mass spectrometry analysis of amphipol-trapped membrane proteins. Anal Chem 84(14):6128–6135. doi: 10.1021/ac301035r CrossRefGoogle Scholar
  2. Boersema PJ, Geiger T, Wisniewski JR, Mann M (2013) Quantification of the N-glycosylated secretome by super-SILAC during breast cancer progression and in human blood samples. Mol Cell Proteomics 12(1):158–171. doi: 10.1074/mcp.M112.023614 CrossRefGoogle Scholar
  3. Charvolin D, Perez JB, Rouviere F, Giusti F, Bazzacco P, Abdine A, Rappaport F, Martinez KL, Popot JL (2009) The use of amphipols as universal molecular adapters to immobilize membrane proteins onto solid supports. Proc Natl Acad Sci USA 106(2):405–410. doi: 10.1073/pnas.0807132106 CrossRefGoogle Scholar
  4. Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372. doi: 10.1038/nbt.1511 CrossRefGoogle Scholar
  5. Giusti F, Rieger J, Catoire LJ, Qian S, Calabrese AN, Watkinson TG, Casiraghi M, Radford SE, Ashcroft AE, Popot JL (2014) Synthesis, characterization and applications of a perdeuterated amphipol. J Membr Biol. doi: 10.1007/s00232-014-9656-x CrossRefGoogle Scholar
  6. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157(1):105–132CrossRefGoogle Scholar
  7. Meissner F, Scheltema RA, Mollenkopf HJ, Mann M (2013) Direct proteomic quantification of the secretome of activated immune cells. Science 340(6131):475–478. doi: 10.1126/science.1232578 CrossRefGoogle Scholar
  8. Ning Z, Seebun D, Hawley B, Chiang CK, Figeys D (2013) From cells to peptides: “one-stop” integrated proteomic processing using amphipols. J Proteome Res 12(3):1512–1519. doi: 10.1021/pr301064z CrossRefGoogle Scholar
  9. Olsen JV, de Godoy LM, Li G, Macek B, Mortensen P, Pesch R, Makarov A, Lange O, Horning S, Mann M (2005) Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 4(12):2010–2021. doi: 10.1074/mcp.T500030-MCP200 CrossRefGoogle Scholar
  10. Polacek M, Bruun JA, Johansen O, Martinez I (2010) Differences in the secretome of cartilage explants and cultured chondrocytes unveiled by SILAC technology. J Orthop Res 28(8):1040–1049. doi: 10.1002/jor.21067 PubMedGoogle Scholar
  11. Polson C, Sarkar P, Incledon B, Raguvaran V, Grant R (2003) Optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography-tandem mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 785(2):263–275CrossRefGoogle Scholar
  12. Popot JL, Althoff T, Bagnard D, Baneres JL, Bazzacco P, Billon-Denis E, Catoire LJ, Champeil P, Charvolin D, Cocco MJ, Cremel G, Dahmane T, de la Maza LM, Ebel C, Gabel F, Giusti F, Gohon Y, Goormaghtigh E, Guittet E, Kleinschmidt JH, Kuhlbrandt W, Le Bon C, Martinez KL, Picard M, Pucci B, Sachs JN, Tribet C, van Heijenoort C, Wien F, Zito F, Zoonens M (2011) Amphipols from A to Z. Annu Rev Biophys 40:379–408. doi: 10.1146/annurev-biophys-042910-155219 CrossRefGoogle Scholar
  13. Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2(8):1896–1906. doi: 10.1038/nprot.2007.261 CrossRefGoogle Scholar
  14. Retz KC, Steele WJ (1977) Acid precipitation of protein in the presence of Triton X-100 and deoxycholate. Anal Biochem 79(1–2):457–461. doi: 10.1016/0003-2697(77)90421-3 CrossRefGoogle Scholar
  15. Salt DJ, Leslie RB, Lillford PJ, Dunnill P (1982) Factors influencing protein structure during acid precipitation: a study of soya proteins. Eur J Appl Microbiol Biotechnol 14(3):144–148. doi: 10.1007/BF00497890 CrossRefGoogle Scholar
  16. Sheng Q, Dai J, Wu Y, Tang H, Zeng R (2012) BuildSummary: using a group-based approach to improve the sensitivity of peptide/protein identification in shotgun proteomics. J Proteome Res 11(3):1494–1502. doi: 10.1021/pr200194p CrossRefGoogle Scholar
  17. Tehei M, Giusti, F, Zaccai, G, Popot J-L (2014) Thermal fluctuations in amphipol A8-35 measured by neutron scattering. J Membr Biol (under review)Google Scholar
  18. Tribet C, Audebert R, Popot JL (1996) Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. Proc Natl Acad Sci USA 93(26):15047–15050CrossRefGoogle Scholar
  19. Wang M, Weiss M, Simonovic M, Haertinger G, Schrimpf SP, Hengartner MO, von Mering C (2012) PaxDb, a database of protein abundance averages across all three domains of life. Mol Cell Proteomics 11(8):492–500. doi: 10.1074/mcp.O111.014704 CrossRefGoogle Scholar
  20. Zoonens M, Catoire LJ, Giusti F, Popot JL (2005) NMR study of a membrane protein in detergent-free aqueous solution. Proc Natl Acad Sci USA 102(25):8893–8898. doi: 10.1073/pnas.0503750102 CrossRefGoogle Scholar

Copyright information

© The Author(s) 2014

Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this license, visit (

Authors and Affiliations

  • Zhibin Ning
    • 1
  • Brett Hawley
    • 1
  • Deeptee Seebun
    • 1
  • Daniel Figeys
    • 1
    Email author
  1. 1.Department of Biochemistry, Immunology and Microbiology, Ottawa Institute of Systems BiologyUniversity of OttawaOttawaCanada

Personalised recommendations