FAQ

  • How to prepare and store my sample(s)

- for protein mass determination ( using ESI-TOF or FT-MS)?
- for protein mix identification with protein in solution
- for protein mix identification with protein separated in SDS-PAGE?
- for protein identification using excised spot from 2D gel?
- for N-term or C-term sequencing using ISD-MALDI-TOF-TOF
- for 2D-DIGE analysis

You have not found the answer to your question or you have other questions, please send a mail to proteomics.giga@ulg.ac.be

 

• How to prepare and store my sample(s) for protein mass determination (using ESI-TOF or FT-MS)?

Every sample to be analyzed should be provided at a concentration of 40-50 µM, in a volume of 50-100 µL, in 25 mM ammonium acetate (NH4Ac) or ammonium bicarbonate (NH4HCO3) buffer. If those buffers are not compatible with short term storage or if you do not want to perform the buffer exchange, sample will be desalted and buffer exchanged on site prior to analysis.

Information on sample purity, expected MW, buffer and other components in solution and sample stability (temperature of storage, possible denaturation/ aggregation in high concentration of acetonitrile) are requested on the analysis request form.

• How to prepare and store my sample(s) for protein mix identification with protein in solution?

A quantity of minimum 10 µg of total protein mix solution is required for protein digestion and identification. The digestion buffer is ammonium bicarbonate, if the sample is in another buffer, a protein precipitation will be done to exchange the buffer and purify the sample.

• How to prepare and store my sample(s) for protein mix identification with protein separated by SDS-PAGE?

The gel of interest could be provided in a few milliliter of water. A scan picture showing clearly the bands of interest to be excised should be provided along with the gel and the analysis request form. The gel should be stored at 5°C.

If you prefer to provide only the band(s) of interest, please excise them with a new and clean razor blade. Avoid trypsin and keratin contaminations also by wearing unpowdered gloves, using media solution prepared with milliQ water filtered with a 45 µm membrane cut-off and by avoiding any contact with skin, hair and any other natural fibers clothing. Bands should be placed in tubes with a few µL of milliQ water to avoid drying and should be stored at 5°C.  Best results are provided using protein quantity visible with Coomassie blue staining procedure.

• How to prepare and store my sample(s) for protein identification using excised spot from 2D gel?

Spot(s) of interest should be provided excised with new and clean razor blade or using automatic spot picker robot. If excision is manually done, avoid trypsin and keratin contaminations also by wearing unpowdered gloves, using media solution prepared with milliQ water filtered with a 45µm membrane cut-off and by avoiding any contact with skin, hair and any other natural fibers clothing.

Samples should be placed in tubes with a few µl of milliQ water to avoid drying and should be stored at 5°C. 

• How to prepare and store my sample(s) for N-term or C-term sequencing using ISD-MALDI-TOF-TOF ?

Sample should be as pure as possible (minimum 95% purity is preferred) and 10 µL of 10 µM protein are necessary.

• How to prepare and store my sample(s) for 2D-DIGE analysis ?

If sample of total or other kinds of protein extracts are prepared for 2D-DIGE, the best is to store them at -80°C. For any other information related to quantification methods and extraction buffer composition, please contact us.

• What about salt contamination? Can you remove salt component from my sample for me?

Yes, we can perform this step for any type of analysis for which it is required. Small additional fees will be charged.

Note that if any sample information concerning buffer and solution composition can not be found, the desalting step will be automatically performed.

• Where to send my sample(s)?

GIGA Proteomics facility :

D. Baiwir, G Mazzucchelli, L. Trzpiot or N. Rosière

Allée du six août 11Bat B6C,

Quartier Agora

4000 Liege Sart Tilman

Belgium

• What is the time to get results?

We guaranty routine analysis to be performed within 10 working days at time of sample delivery on the Facility site. For other analysis, not considered as routine and long term project the delay would be specified on request in the quotation.

• Under which format will the analysis result(s) be communicated?

Results are communicated by e-mail or via a secured server download link. The report (pdf file) will encompass all experimental details and picture(s) of mass spectra if required. The raw data files may also be send on special request.

• What is the preferred method of coloration for protein identification?

The preferred coloration method which can guaranty protein identification is Coomassie blue.

• What is the limit of detection of general methods of coloration available on the proteomic platform?

Coomassie blue:  50-100ng

Silver staining compatible with mass spectrometry: 0.5-10ng

Syproruby: 0.25-1ng

Cy- Dye: 125pg

• What is the average number of protein identified in a mix of protein by LC/MS-MS?

This depends on your sample complexity and on the proportion of every protein composing the mix, but we observed more than 15 proteins in one experimental run for a protein mix isolated from a Coomassie blue stained 1D-gel band.

• What is the limit of detection for protein identification by LC/MSMS?

Protein(s) identification was obtained with a very weak band isolated from a 1D gel stained with Sypro-Ruby.

• Is the classical N-terminal Edman degradation technique better than ISD-MALDI sequencing?

It is neither better, nor worse! The quantity of material needed is different and the information provided by both techniques is complementary. N-terminal and C-terminal sequencing by ISD –MALDI-TOF-MS provide a sequence from the 10th to the 40th amino acid (The entire sequence can be achieved if the sample material enables it, but this needs to be experimentally determined.) The N-term Edman degradation sequencing provides information on the first 15 to30 amino acids.

• Is it possible to verify the efficiency of my protein labeling by mass spectrometry?

Yes, but the choice of the technique will depend on the type of label you target (chemical tags, biotin, streptavidin…), the protein purity and the mass range you are interested in. However, the analysis will be semi-quantitative if the mass determination and comparison of labeled and unlabeled sample is performed using ESI-TOF, FT-MS or MALDI-TOF-TOF.

Please ask for personalized solution.

• What are the prices?

All prices are on request and special discount might be applied if a high number of analyses is requested.

• Where to find submission forms for my analysis?

You can find analysis request form in the services section. Please refer to the desired service to find the right analysis request form.

• What is the mass accuracy for FT-ICR-MS mass determination?

Below 1 ppm in the mass range 80Da – 1000Da, the results of the calibration of the day is always communicated within the analysis report.

• Is it possible to obtain elemental composition for my compound?

Yes, this analysis is possible using FT-ICR-MS analysis for molecules with a molecular weight below1000Da.

• What is the mass accuracy for ESI-TOF-MS mass determination?

Mass accuracy for ESI-TOF mass determination is around 100ppm.

• Is my method of coloration suitable for mass spectrometry analysis?

All classical coloration methods not involving any cross linking step of colorant or other component to protein are compatible. Please contact us if you have any doubt!

• What is the water quality grade necessary for mass spectrometry analysis?

Fresh Milli-Q water, filtered using a 45µm membrane cut-off should be preferred. Please do not store water and sample in plastic containers as polymers contamination could occur.

• Which contamination needs to be avoided for mass spectrometry measurement?

Contaminations with salts, urea, thiourea, detergents, plastic polymers, glycerol, PEG and other cryoprotectants need to be avoided.

• What type of information is necessary for databases searches for protein(s) identification?

The phylum and species are the best information to provide and if the sequence of the source organism is not completely known, the closest organism already encoded in the databases is the best. If nothing is specified, searches will be done against all taxonomies.

If the sequence of a synthetic protein should be retrieved, this could be introduced in a private in-house database which stays confidential to any other parts.

• Can you identify a protein from an organism for which the genome is not or not yet completely known?

Yes, in general searches on the databases are able to deal with this problem and find similarities with the closest organisms known and encoded to date.

• Can you identify a protein which has not yet been observed as a true protein: meaning hypothetical protein(s) or EST?

This is possible by using specific databases (MDSB and NCBI) containing information deduced from genome sequence, including EST and hypothetical protein deduced by similarity searches.

• Is possible to get what is left of my sample after analysis?

Yes, any remaining of the analyzed samples is stored on site for 3 years according to our quality system. On request, you can get back the remaining sample(s).

• What about confidentiality on my sample, analysis and results?

All analysis, contacts, information provided by our costumers so as the results of analysis are kept confidential.

• Is it possible to analyze a synthetic peptide that was “in vitro”, chemically modified?

This may be performed but depending on sample concentration, material availability and purity, the technique must be carefully chosen.  Please contact us for a personalized solution and quotation.

• Is it possible to study “protein interactome” using the proteomic techniques available on the platform?

This is possible, but the choice of the technique depends on your aim and experimental context, thus please contact us for personalized solution.