While Proteomic Platform tries to adapt its responses to each sample and each problematic issues, here are some common questions and answers.
What facilities does the Plateforme Protéome propose ?
The Plateforme Protéome gives the scientific community of Region Aquitaine an opportunity to at a low cost make use of recent developments in techniques and instrumentations for the analysis and identification of proteins. The facility is equipped with 2-D electrophoresis systems, FPLC chromatography, MALDI-TOF and MS/MS instruments (cf Scientific Equipment). The facility has expertise for protein purification, 2-D analysis, mass spectrometry, data base searches and general protein chemistry.
Our area of work is analysis and comparison of proteomes, identification of proteins in protein spots/bands by mass spectrometry, analysis of expressed proteins for quality control and analysis of proteins for post-translational modifications. The facility is open for all scales of problem solving or analysis, although the capacity for 2D gels might be a limiting factor for very large undertakings. The facility has in addition a limited capacity for de novo sequencing of proteins from any species.
Under which form does these facilities are proposed ?
Several levels are possible. Submitted samples can be entirely treated by the Plateforme Protéome team. Otherwise, users can join the team in order to acquire autonomy necessary to realize the project. Our role consists in providing researchers protocols and expertise.
What minimal amount of protein leads to an identification ?
We generally consider that that protein has to be visualized on gel. Nevertheless, if a Coomassie stain protein (~0.1 µg) is generally identified, the Plateforme Protéome can not guarantee identification when more sensitive staining procedure are used. Indeed, in such a case, staining becomes « protein-dependent »: some proteins are identified and others are not. One must keep in mind that identification confidence increase with protein amount. This problem of quantity is critic when one wants to cover the whole sequence of a protein, particularly when post-translational modifications are searched.
Which staining procedure is preferred ?
As previously discussed, the staining protocol leading to best identification is Coomassie Blue. Most staining procedures still are compatible with mass spectrometry at the exception of silver stains that includes a fixing step in glutaraldehyde or formaldehyde at sensitization or staining steps.
How pure does my sample has to be for a MS analysis ?
While Peptide Mass Fingerprint is limited to the analysis of isolated protein, LC-MS/MS is better suited to the analysis of complex protein mixture. Nevertheless, a poorly represented protein in a complex mixture might be not identified. In such a case, it is strongly recommended to deplete major proteins such as Ig, actin, tubulin, albumin … Keratin contaminations must be avoided for the same reasons.
Others contaminant such as salts and detergents are to be avoided or kept under concentration threshold given somewhere else.
Can mass spectrometry sequence a protein ?
The classic proteomic strategy consists, through MS experiments, in obtaining sequence informations and not the sequence itself. Nevertheless, it remains possible to perform « de novo » sequencing on some peptides so as to extract short sequence “tags”. The fine analysis of MS/MS spectra leads to the obtaining of short peptide sequencing thus enabling the construction of nucleotidic probes.
What recommendations are to be followed for cutting gel pieces ?
Basic rules for avoiding keratin contaminations are exposed elsewhere (« Keratin » contaminations). Briefly, the gel and everything that might contact it have to be kept away from dust, skin and hair. It is also recommended to maximise the protein/acrylamide ratio.
How samples have to be submitted ?
The Plateforme Protéome accepts sample under a variety of conditioning (whole gel, gel pieces, solutions …) as soon as these samples have been treated under rules preventing it from keratin contamination (see above). Polyacrylamide gels must not be dried but stored in Acetic Acid 1%.
How to understand the results of mass spectrometry from Proteome Discover ?
You can find this document on excel explanations of each column: Support Document
The entire staff of the Proteomics Platform is at your disposal to answer your questions and help you in understanding different attachments to the final report. On your request, a review meeting will be organized