Dunn School | CPF (Headington) | CBRG | University of Oxford

Dunn School of Pathology

Central Proteomics Facility

Methods - This area provides information and links on a range of proteomics methods that may be relevant to your research.

Gel Band ID - The most basic type of proteomics experiment involves identifying a gel band. Make sure you use a mass spec compatible staining mathod such as Sypro Ruby, coomassie or a mass spec compatible silver stain. Invitrogen and Sigma make mass spec compatible silver stain kits. Use the highest grade reagents available and try and avoid keratin contamination. We use Invitrogen's NuPage precast gels, running buffer and loading dye in order to minimise keratin contamination. Protocols and advice are available in the protocols section.

Complex Protein Mixtures - Modern proteomics instrumentation and methods are capable of analysing and identifying 10s, 100s and even 1000+ proteins present in a complex mixture. More starting material, more sample prefractionation and more mass spec time will result in increased numbers of identifications.

GeLCMS - A complex mixture is run on a mini SDS PAGE gel. The enitire gel lane is then cut into 10 equal gel slices, digested in-gel with trypsin and then analysed by LC MS/MS or LC MALDI

SCX - a complex mixture is first digested in-solution with trypsin. The peptides are then prefractionated using strong cation exchange (SCX) chromatography. Each SCX fraction is then analysed by LC MS/MS or LC MALDI.

Protein Complexes - A number of techniques exist for identifying components of protein complexes.

• TAP Tagging - Tandem Affinity Purification generally produces clean material with minimum background. Optimisation is generally required in order to produce sufficient material for mass spec analysis.

  Some useful TAP taf links and references.

  mammalian TAP tagging protocol

  Ref:Tsai A, Carstens RP An optimized protocol for protein purification in cultured mammalian cells using a tandem affinity purification approach.
  Nat Protoc. 2006;1(6):2820-7.

  Ref:Burckstummer T, Bennett KL, Preradovic A, Schutze G, Hantschel O, Superti-Furga G, Bauch A. An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells.
  Nat Methods. 2006 Dec;3(12):1013-9. Epub 2006 Oct 22.

  Ref: Drakas R, Prisco M, Baserga R. A modified tandem affinity purification tag technique for the purification of protein complexes in mammalian cells.
  Proteomics. 2005 Jan;5(1):132-7.

  Ref:Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, Seraphin B. A generic protein purification method for protein complex characterization and proteome exploration.
  Nat Biotechnol. 1999 Oct;17(10):1030-2.

  
  

• Biotin based tags - high affinity methods capable of producing large amounts of material. Biotinylation of proteins other than the bait can prove problematic. Expressing the bait and the biotin ligase is also problematic.

  Some useful links on biotinylation tagging systems

  Ref:de Boer E, Rodriguez P, Bonte E, Krijgsveld J, Katsantoni E, Heck A, Grosveld F, Strouboulis J. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice.
  Proc Natl Acad Sci U S A. 2003 Jun 24;100(13):7480-5. Epub 2003 Jun 11.

  Ref: Rodriguez P, Braun H, Kolodziej KE, de Boer E, Campbell J, Bonte E, Grosveld F, Philipsen S, Strouboulis J. Isolation of transcription factor complexes by in vivo biotinylation tagging and direct binding to streptavidin beads.
  Methods Mol Biol. 2006;338:305-23.




• Quantitative IPs QUICK - SILAC based IP strategy to distinguish non-specific from specific interacting partners.



• Flag tag - useful system, but can suffer from non-specific background arising from anti-flag ABs.



• Strep tag - a short, very high-affinity tagging system, producing abundant amounts of material. See manufacturer's website for more information.

Quantitative Proteomics - A number of proteomics techiques exist for investigating the amount of protein present in a sample. Broadly, these can be separated into relative quantitation techniques and absolute quantitation techniques.

• SILAC

Stable Isotope Labelling by Amino Acids in Cell Culture (Recent ref: A Practical Recipe for Stable Isotope Labelling by Amino Acids in Cell Culture, Nature Protocols, 2006; 1 (6): 2650-60) is a relative quantitation technique in which one cell culture is grown in the presence of a light-label amino acid and the other in the presence of a heavy-label amino acid. The mass spectrometer and software are then used to determine relative expression levels of protein in the two samples. SILAC kits are available in kit form from Invitrogen, though it may be cheaper to buy your own reagents and media - contact us for advice.

Useful links

http://www.silac.org/

http://www.pil.sdu.dk/silac_intro.htm

http://www.invitrogen.com/content.cfm?pageid=10862

http://msquant.sourceforge.net/

• ITRAQ

A relative proteomics labelling technique using post-labelling with isotopically labelled tags. The technique allows 4 samples to be compared simultaneously (4-plex), but will shortly support 8-plex quantitation. ITRAQ kits can be purchased from Invitrogen.The 4800 MALDI TOF TOF supports ITRAQ.

• AQUA

A method of determining the absolute amount of protein present in a sample. A known amount of an isotopically labelled peptide analogue is doped into the sample and used to quantitate against (Reference: Gerber, Scott A, et al. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. PNAS. June 10, 2003. Vol 100. No 12. p 6940-6945). Cutom AQUA peptides are available from Sigma. The technique has applications in quantifying post-translational modifications and biomarkers.

• Label Free Quantitation

Label free quantitation methods are gaining in popularity. The technique does not use isotopic labelling of proteins, but instead directly compares signal intensities between different LC MSMS runs. The Orbitrap is equipped with SEIVE software capable of label free quantitation.

• Other Techniques

A large number of other methods exist for quantitation - too numerous to list exhaustively. See review by by Goshe and Smith

ICPL - isotope coded protein labelling

Metabolic labelling with 15-N

labelling with 18-O heavy water

ICAT - isotope coded affinity tag (cysteine tagging)

Post Translational Modifications - Both the Orbitrap and the 4800 MALDI TOF TOF are capable of identifying the nature and position of post translational modifications. the exact nature of the experiment will depend on the type of post translational modification.

links to useful websites and references on various PTMs.

• phosphorylation


• Quantitation of phosphorylation sites.



• Titanium dioxide phosphopeptide purification protocol.

Biomarkers - Biomarkers present in tissue, serum, plasma etc can be quantitated and identified using various approaches. Post-labelling techniques such as ITRAQ are suitable for analysing a small number of samples in great depth, but are unsuitable for analysing large sample cohorts due to the amount of time required. When used in conjunction with sample prefractionation and MarkerView software the 4800 is capable of analysing a large number of samples for differences

A number of prefractionation media exist.

• Bruker ClinProt magnetic beads. Scaleable and available in a wide range of different media.


• Perkin Elmer proXPRESSION enrichment system. Membrane based purification system.


• Ciphergen's/Bio-Rad's SELDI (Surface Enhanced Laser Desorption/Ionisation) target plates.

Please contact us to see if we can help.

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