Sample preparation guidelines
Inappropriate sample handling is the most common source for contamination and other problems in the downstream analysis. Please read the following considerations carefully and keep them in mind when handling your samples and gels – the quality of your data depends on it.
- Contaminations can occur in all phases of sample handling.
- Dust particles contain high levels of proteinaceous contaminations (hair and skin particles, wool fibers, etc.) and as a consequence you should try to work as cleanly as possible. Wash or clean all equipment very thoroughly before use, filter solutions, wear (powder free nitrile) gloves and lab coats at each step, use chemicals and solutions dedicated to "mass spectrometry", use new pipette tips instead of autoclaved ones, and do not touch the vials at any stage of the sample preparation without gloves.
- Low quality plasticware or solvents stored in plastic containers can severely contaminate your sample with plasticizers (polymers), impede the analysis or make it completely impossible. Contact us if you are in doubt of what to use.
- Bear in mind that “clean” glassware which comes directly from the dishwasher may contain considerable amounts of detergents.
- In case you are not sure about the "cleanliness" of your laboratory, it is possible to use our laminar flow for particular preparation steps (e.g. gel band cutting). Please contact us in advance so that we can arrange the hood for you or help you with your setup.
Further, method specific points to consider
1. Proteins separated by gel electrophoresis
There are several methods to visualize proteins after gel electrophoresis. Unfortunately, not all of them are compatible with mass spectrometry. Coomassie staining techniques are in general ideal. However, there are some cases when higher sensitivity is needed. Please note that silver staining applying glutaraldehyde in the sensitizing solution is not compatible with mass spectrometry. For this reason we recommend using Coomassie staining or an alternative silver staining protocol (link). In case of silver staining do not overstain the gel, keep the background clear. In our experience, overstaining reduces the yield of identifiable peptides from the sample substantially.
Carry out all staining steps in a clean and closed container and cut the bands/spots under a laminar flow hood. Do not use containers that have been used for Western blocking before (milk powder, etc.) and only use clean (filtered) buffers.
Bands or spots should be cut into dices of approximately 1 mm3 with a clean scalpel or razor blade and stored in a clean and labeled tube.
Keep the volume of the empty gel matrix as small as possible, cut away unstained gel material.
Submit the samples as soon as possible; freeze them only if you can guarantee that they will not thaw during the transfer to our lab. If you send the sample via mail, please contact us before and send it on dry ice.
2. Protein solutions or pellets
Please note, that samples should not contain high amounts of salts (> 100 mM), should be free of non-volatile buffer components (e.g. phosphate buffer), ionic detergents (e.g. SDS, Triton, etc.), glycerol, polymers such as PEG, and radioactive labels.
Use detergents very carefully during sample preparation, they cannot be removed by dialysis and only some of them can be removed by other means but often with considerable loss of sample. In all cases please let us know which detergents you used and how, so that we can see if we have to do something about it. Injecting detergents into an LC-MS system does not only hamper the current analysis but may severely reduce overall performance requiring long and costly cleaning procedures.
Fresh protein solutions should be delivered on ice as soon as possible or snap frozen in liquid nitrogen and delivered on dry ice. If you send the sample via mail, please contact us before and send it on enough dry ice to prevent thawing (avoid sending over the weekend and public holidays and take into account customs regulations if you send it from abroad).
3. Proteins bound to beads
If you isolate your proteins with beads coated with proteins (e.g. antibodies, calmodulin, etc.), please contact us before you submit the sample. If the proteins are bound to beads, please submit the samples in a fresh state, do not freeze them! Please also read through the considerations for protein solutions above as they similarly apply to bead samples.
4. Samples for cross-linking mass spectrometry (XL-MS)
MFPL users can have a look at our detailed instructions here.
5. Requirements for intact protein mass analysis
- Min. 10 µL of 0.5-1 mg/mL purified protein(s)(or 0.1-1 pmol as a rough guideline; we do inject less eventually but prefer to have some more sample as a buffer)
- Standard biological buffers such as TRIS, HEPES or PBS are compatible.
- Glycerol < 5%, most salts, and reducing agents are acceptable. Nevertheless, try to keep the concentration of all these agents as low as possible (especially salts).
- NO detergents (SDS, Triton, etc.) and stabilizers such as PEG!
- When the protein is lyophilized, reconstitute it in 5% organic solution (acetonitrile or methanol) with 0.1% formic or acetic acid or submit the dry sample and we will do this for you.
- Samples will be reduced by default, unless you specify a reason against it.