FAQs

I would like to submit a sample for analysis. What do I need to do?

First, read the instructions . Then, please send a short description of your analytical project by e-mail, schedule a meeting with Dr. Marc Moniatte to discuss about your experimental goals and eventually clarify the sample preparation before beginning your experiments to be analyzed by mass spectrometry. It is a good idea to provide us with an electronic version of the AA sequence if you know the protein and with a scanned image of the gel if the protein(s) you want to analyze were separated so. Do not forget to print the submission form out and bring it with you when you drop off your samples.

Where are you located?

The PCF-PTP office is located at the West end of the EPFL campus in the extension of the AI building (near the SV Building).

Do I need to make an appointment to drop off a sample?

Making an appointment is usually not necessary if you have briefly discussed about your analytical project before. The person in charge of your samples will have agreed with you on a date. Should you have issues with your preparation, need more information or simply want to make sure that your sample will be taken care of, please email or phone call one of us to schedule an appropriate meeting.

I would like to qualitatively map some phosphorylation sites of my immunoprecipitated protein. How pure does the sample have to be and how much material do I need?

In most cases, phosphorylated peptides are present at a low stoichiometry compared to their non-phosphorylated counterpart. In addition to that their MS signal in positive ion mode is often very low or suppressed by abundant non-phosphorylated peptides. This is the reason why it is absolutely necessary to enrich phosphopeptides prior to LC-MS/MS analysis. At the facility we have selected the MOAC approach with TiO2 as method of choice. The method has been optimized to be very selective so phosphopeptides in a mixture can be detected and analyzed with reasonable chances of success. The accurate localization depends on the quality of the tandem-MS spectrum so if you want to maximize the chances of detecting and localizing the modification(s) we advise you (whenever possible) to provide 10-15 µg of your protein of interest if the protein is pure. More material might be necessary if the protein is at low concentration in a mixture or if the stoichiometry is expected to be low.

In what buffer can I submit my sample?

Please, follow these detailed recommendations . For samples submitted in solution, we prefer diluted? MS compatible buffers like ammonium bicarbonate (AmBic or AB) or Tris buffers (50-100 mM).  Wrong pH, detergents, organic solvents and salts are the main causes for failed MS measurements. We firmly recommend you to provide us with a detailed description of  the solution composition in which   your sample will be submitted (Organic solvent? Detergents? Buffer? pH?…). This step is critical because we have different protocols available for the removal of some incompatible buffer/detergent (Desalting, FASP…). For samples requiring special pH, detergents or salts to overcome solubility issues it is often possible to replace them by MS compatible formulations. Classical detergents (SDS, NP40,…) can advantageously be replaced  by acid labile detergents (Waters RapiGest, PPS,…) or MS tolerant detergents like n-octylglucopyranoside or deoxycholate. High pH buffers can be neutralized and salts reduced or removed by simple sample pretreatment provided that we know what is in the solution.  Do not hesitate to contact us if you are not sure.

What is the expected turn-around time for routine analysis? (protein identification from a 1D gel band, sample in solution, analysis of interacting proteins from an IP, …)

The time needed to deliver the results is dependent on the workflow employed and the laboratory workload at the time of sample delivery. Usual turnaround time remains between one and two weeks from sample deposition to result delivery for small protein identification projects (up to 8 samples). Projects involving larger number of equivalent samples (gel bands) may take longer and are depending on the laboratory workload. These projects are anyway subjected to timeline projection and will be evaluated on a case-by-case basis with you when you bring your samples.

What results can I expect in terms of phosphorylation site localization accuracy from a phosphopeptide enrichment experiment like the one you propose?

Please read a description of this service . Briefly, proteins are usually digested after a reduction alkylation step. The resulting peptide mixture is enriched in phosphopeptides by metal affinity chromatography which are then analysed by rpHPLC-MS/MS. The expected result depends on the quality of the tandem-MS spectrum (the number of informative fragments) which depends on the amino-acid sequence and on the amount of peptide on the column.
This analysis can lead to the identification of phosphopeptides for which the localization of the phosphorylation site is unambiguous. This is usually the case for peptides containing only one modifiable residue on the peptide (monophosphorylated peptides) or when the number of phosphate moieties equals the number of modifiable residues (di- or tri-phosphorylated peptides). In case of ambiguity on the localization, the output of different algorithms is combined into a statistical confidence interval for the localization of the phosphate moiety on the peptide (see here for any detail).
 

I want to sequence my protein. Do you do Edman Sequencing ?

The PCF-PTP is not equipped for this type of requests. We can recommend our colleagues at the FGCZ in Zurich who seem to be the only ones still offering this service.

I want to separate a complex brain protein extract and get relative quantification of the proteins. Does the facility provide 2D-Gel DIGE service ?

The PCF-PTP is not equipped for this type of requests. The demand is too irregular to maintain this service. We have a standing agreement with another facility at the CIG in Lausanne that offers 2D-Gel and 2D-Gel DIGE service since many years. Please contact Dr. Manfredo Quadroni at the Protein Analysis Facility (Lausanne)

What is SILAC?

SILAC is an elegant way to quantify changes in protein abundance. As suggested by the name, SILAC (Stable Isotope Labeling with Amino acids in Cell culture) is a labeling method based on stable isotopes (non radioactive) which can provide accurate (relative) quantitative information on proteins from two cell populations. (heavy amino acids are non radioactive and can be used in every cell culture lab.) In a typical SILAC experiment, proteins are labeled using depleted cell culture media supplemented with either normal isotope abundance (light) or stable isotope labeled (heavy) amino acids.. After digestion, proteins are observable in the same mass spectrum and distinguishable by their respective “light” and “heavy” peptide MS signals. When samples are mixed in equal proportions, MS signals from both populations are detectable unless the absence of either the light or heavy member is a direct result of the experimental perturbation. As labeling is taking place very early (at the protein level) SILAC enables complex fractionation/enrichments steps without being spoiled irremediably by experimental variation typical of these pipelines. The facility has already experience in pair wise relative quantification of protein extracts, specific protein-protein interaction determination and PTM abundance relative quantification using this technique.

I have detected my protein by Western Blot. Can you identify my protein and confirm that result by MS?

On a classical western blot, you can have signal from hundred of molecules because you amplify your signal (Secondary antibody linked to horsedish-peroxidase cleave your chemiluminescent agent). This Western blot approach only allows you to see your protein of interest. MS is often less sensitive to this technique because the presence of abundant proteins (actin, tubulin, metabolic enzymes …) can mask the detection of peptides coming from your protein of interest. Nevertheless, if you want to analyze your protein of interest and or its interacting partners you can design a purification procedure or easily perform immumoprecipitations.

I am only interested in my protein. Is it possible to monitor specific proteins or peptides in a complex sample?

If your protein is a minor component within a very complex mixture, it’s necessary to use specialized MS approaches to target your protein. If you know the sequence of your protein, we can target the analysis of peptide ion masses expected to be generated from your protein sequence.

How much sample amount do I need to submit for protein identification? What is the sensitivity? Do I have to see a band for my protein of interest?

There is not a clear rule for all the proteins because they are quite heterogeneous in their amino-acid and PTM compositions. For example, transmembrane proteins can be sometimes quite difficult to digest and analyze. We prefer to work on gel bands visible on Coomassie-stained gel but we can identify proteins coming from silver/sypro-stained band. For in-solution digestion and analysis, we ask 10ug for LCMS if possible. If you want to have a good result, it’s always better to spend some time to prepare a sufficient amount of sample instead of having to repeat the analysis several times.