Large scale protein quantitation
The heart of this approach is the simultaneous separation, identification and quantitation of thousand of polypeptides. The Geneva Proteomics Core Facility provides 3 large-scale protein quantitation services to which you may send your samples for analysis. These services include:
- SILAC + 1-DE + LC ESI-MS/MS:
We propose to use the SILAC strategy, which is specifically designed for the quantitative and qualitative study of proteomes of cultured cells. For this purpose, two cell populations, derived from a single cell line, are cultured either in normal conditions using natural isotopic reagents or in absence of one essential amino acid, which is replaced by an isotopically heavy form. Each cell population is then exposed to different stimuli. The cells of the two populations are subsequently combined and their proteins identified by MS/MS. The peptides containing both normal and heavy forms of the amino acid can be easily distinguished by a mass spectrometer. In addition, the ion intensity ratio between normal and heavy peptides provides quantitative information, which allow to highlight the proteins whose abundance or modification have been altered.
- Isobaric tagging + offgel electrophoresis + LC ESI-MS/MS:
We propose the recently introduced isobaric tagging technology for simultaneous quantification by mass spectrometry. Several products have been set as multiplexed reagents that can label all peptides in up to six, eight or ten different biological samples enabling simultaneous identification and quantitation, both relative and absolute, while retaining important PTM information. Labelled isobaric peptides, which have the same mass in MS, make interpretation easier in MS spectra. In MS/MS, these labelled peptides fragment, generating signature ions that enable quantitation in the low mass region. The main advantages of this approach over 2-DE proteomics strategies:
- Increase protein and proteome coverage by labelling all peptides, including those with post-translational modifications.
- Boost assertion in identification and quantitation from MS/MS spectra by tagging multiple peptides per protein.
- Endow with the flexibility to duplex, triplex, or multiplex up to six different biological samples simultaneously in a single experiment.
- 2-DE gels + Sypro Ruby + Image analysis:
We propose to run 2-DE gels using 3.5-10 or 4-7 IPG strips to display a wide range of proteins. However, the use of 1 pH unit narrow range IPG is possible. Today, silver staining is probably the most popular non-radioactive protein detection as it is more sensitive than Coomassie Brilliant Blue or reverse staining. We also propose to stain the gels with fluorescent staining. Image analysis is performed using the Image Master software in collaboration with the Geneva Bioinformatics Core Facility.
Sample preparation and solubilization are crucial factors for the overall performance of the 2-D PAGE technique. Protein complexes and aggregates should be completely broken up in order to avoid the appearance of new spots due to a partial protein solubilization. Thus, samples have to be sent to the Geneva Proteomics Core Facility either in a fresh solution containing 8 M urea, 4% (w/v) CHAPS, Tris base 40 mM, DTE 65 mM and a trace of bromophenol blue (see below) or as a dried powder. The minimum amount of protein has to be around 100 µg. The sample may contain salt in amounts lower than 100 mM.
Put your samples in an Eppendorf tube, freeze them at -20 degree Celsius and put them into dry ice for shipment.
|Method||Swiss Fr. / sample|
|SILAC + 1-DE + LC ESI-MS/MS||Inquire|
|Isobaric tagging + offgel electrophoresis + LC ESI-MS/MS||Inquire|
|2-DE gels + Sypro Ruby + Image analysis||Inquire|
Allow six weeks for delivery of the results.