Proteomics pp 61-67 | Cite as

Full Membrane Protein Coverage Digestion and Quantitative Bottom-Up Mass Spectrometry Proteomics

  • Joseph Capri
  • Julian P. WhiteleggeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1550)


A true and accurate bottom-up global proteomic measurement will only be achieved when all proteins in a sample can be digested efficiently and at least some peptides recovered on which to base an estimate of abundance. Integral membrane proteins make up around one-third of the proteome and require specialized protocols if they are to be successfully solubilized for efficient digestion by the enzymes used in bottom-up proteomics. The protocol described relies upon solubilization using the detergents sodium deoxycholate and lauryl sarcosine with heating to 95 °C. A subset of peptides is purified by reverse-phase solid-phase extraction and fractionated by strong-cation exchange prior to nano-liquid chromatography with data-dependent tandem mass spectrometry. For quantitative proteomics experiments a protocol is described for stable-isotope coding of peptides using dimethylation of primary amines allowing for three-way sample multiplexing.

Key words

Trypsin Electrospray ionization Proteome StageTip Dimethylation Phase transfer 


  1. 1.
    Ryan CM, Souda P et al (2010) Post-translational modifications of integral membrane proteins resolved by top-down Fourier transform mass spectrometry with collisionally activated dissociation. Mol Cell Proteomics 9:791–803CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Whitelegge JP (2013) Integral membrane proteins and bilayer proteomics. Anal Chem 85:2558–2568CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Manza LL, Stamer SL et al (2005) Sample preparation and digestion for proteomic analyses using spin filters. Proteomics 5:1742–1745CrossRefPubMedGoogle Scholar
  4. 4.
    Wiśniewski JR, Zougman A, Mann M (2009) Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. J Proteome Res 8:5674–5678CrossRefPubMedGoogle Scholar
  5. 5.
    Masuda T, Tomita M, Ishihama Y (2008) Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J Proteome Res 7:731–740CrossRefPubMedGoogle Scholar
  6. 6.
    Erde J, Loo RR, Loo JA (2014) Enhanced FASP (eFASP) to increase proteome coverage and sample recovery for quantitative proteomic experiments. J Proteome Res 13:1885–1895CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kulak NA, Pichler G et al (2014) Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat Methods 11:319–324CrossRefPubMedGoogle Scholar
  8. 8.
    León IR, Schwämmle V et al (2013) Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis. Mol Cell Proteomics 12:2992–3005CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906CrossRefPubMedGoogle Scholar
  10. 10.
    Wilson-Grady JT, Haas W, Gygi SP (2013) Quantitative comparison of the fasted and re-fed mouse liver phosphoproteomes using lower pH reductive dimethylation. Methods 61:277–286CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of PharmacologyDavid Geffen School of Medicine, UCLALos AngelesUSA
  2. 2.The Pasarow Mass Spectrometry LaboratoryThe Jane and Terry Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, UCLALos AngelesUSA

Personalised recommendations