Very Frequently Asked Questions
MS , protein identification and PTM's
Q : What sort of sample is accepted for protein identification by MS ?
A : Polyacrylamide gel slices containing the protein of interest are accepted. Both samples from 2D- and 1D SDS-PAGE gels can be accepted. Of course, on 1D gels often protein bands overlap.
Q : Are proteins in solution accepted for mass spec ?
A: Since it is difficult to know the exact composition of liquid samples, proteins in solution are usually not accepted. Contact us to discuss your particular case.
Q : Do I have to be able to see a band for my protein of interest ?
A : Yes, this is the most important prerequisite. If you do not see a definite band by some protein detection method, you probably won't get any useful data.
Q : What is the sensitivity ? How much protein do I need ?
A : The more, the better ! If you want to have a good result you should plan to load on your gel about 1 pmol of the protein you want to analyse. For a polypeptide of 50 kDa this corresponds to about 0.05 µg. It is possible to obtain results with less (down to 100 fmol starting material) but then the nature of the protein tends to play a major role. Some proteins just digest and are recovered much better than others. Remember, it is worth investing a bit more work to prepare 2-3 times more protein than having to repeat the analysis over and over.
Q : How pure does my protein have to be ?
A : Again, as pure as you can get it. Remember, mass spectrometry is a physical technique, so it will detect ANY polypeptide material. Most biological samples purified from in vivo material contain relevant amounts of highly abundant cellular proteins such as actin, tubulin, or metabolic enzymes ...so you have to design a purification procedure that can resolve your (usually low abundance) protein from these ones.
Q : My sample looks pure by western blot. Can I submit it for analysis ?
A : No. Even if you have only one clean band by western, you probably have hundreds of proteins in your sample. Try to silver stain a 1D gel of your sample and find out if there is a band that could correspond to your protein.
Q: How should I prepare my sample ?
A : Run your gel, stain with Coomassie Blue, silver stain or a fluorescent dye (e.g. SYPRO ruby).
Cut under clean conditions the band you want to analyse, put it in an Eppendorf and bring it to us.
Q : Do I have to take special precautions when preparing my sample ?
A : Try to work in clean conditions. The most frequent ubiquitous contaminant is keratin, that covers virtually any object used by humans. Keratin is actually one of the major components of common DUST. Wash well your gel plates. Wear gloves when loading your gels and especially when cutting the band or spot. If possible, do the cutting under a laminar air flow (hood). Use clean Eppendorf tubes.
Q : What staining protocol can I use ?
A : All most common staining methods are compatible with subsequent analysis. Avoid any fixing step employing glutaraldehyde or paraformaldehyde. In general the less you fix, the better (but this is often a trade-off with staining quality). Colloidal Coomassie Blue or rapid Silver staining (see under protocols) usually give the best results.
Or, if you can afford it, the fluorescent Sypro Ruby stain.
Q: How big can the gel slice be ?
A: The best way to excise proteins is to use a pipette tip that has been cut so to give an aperture of 1.0-1.5 mm
in diameter and punch out a round gel piece. We both use Pasteur pipettes or plastic ones for this.
Make sure the gel is well hydrated when you cut. Cutting the gel "under water" is a good idea, it also helps reducing keratin contamination. Cutting can be a delicate procedure and you should practice on a test gel before going for a real sample.
Q: My protein is spread over a large surface. Should I cut it all ?
A : Unfortunately protein concentration in the gel is a critical factor for the efficiency of the digestion. 0.1 ug of
protein on a 1 mm2 surface will give a better yield than 0.5 ug on 20 mm2. In addition, processing large gel volumes
poses a number of technical problems. Therefore, try to maximize protein concentration and only cut the "darkest" portion of your silver or Coomassie spot.