To achieve optimal results from the mass spectrometric analysis of your samples please read and follow carefully the following guidelines for sample preparation and submission.
General rules for sample submission
We strongly suggest that you discuss the general strategy of your bioanalytical question with either Karl Mechtler or another qualified person before you generate the samples.
Before you bring your samples to the protein chemistry facility, you must contact Karl Mechtler (IMBA Plaza floor, room number 5.11, tel: 4280) to discuss your analytic question. In case Karl Mechtler is not available, please contact Elisabeth Roitinger (IMBA Plaza floor, room number 5.44, tel: 4298). For the actual submission of your samples please download the sample submission form from the "Protocols" section and follow the mentioned steps. Send the completed form to proteomics(at)imp.ac.at.
Sample preparation guidelines for proteins in solution
Use MS-compatible buffers
Samples should not contain high amounts of salts (>100 mM), should be free of non-volatile buffer components (e.g. phosphate buffer), free of protease inhibitors and free of detergents (e.g. SDS, Triton, Tween, NP40). Additionally, the sample volume should be as small as possible (approx. 20 - 100 µl) and the pH of the sample should be around 7-8. Use 1M TRIS buffer (pH 8.0) to neutralize your sample if necessary (you can get this buffer in the media kitchen).
We also provide a protocol for protein immunoprecipitations (IP), which is compatible with MS.
If you want iTRAQ-based quantification of your proteins do NOT use TRIS buffer but 1M TEAB instead (please get this buffer from our laboratory). In this case also other primary or secondary amines must be absent for a successful analysis.
Avoid using polyethylene glycol containing detergents
PEG-containing detergents such as Triton, Tween or NP40 present in your sample will dramatically reduce the quality of your data. Even trace amounts of these detergents will be a problem during the MS-analysis. If you have to use them during an IP-experiment, you will need to wash the resin very well and if possible transfer the resin to a fresh tube. Even, if you are not using these detergents yourself, be aware that lots of things in your lab are probably contaminated with PEG containing detergents, such as:
o Glassware previously used for buffers
o Glassware washed in a dishwasher
o Pipettes could be contaminated
o Communal lab chemicals
o Organic solvents stored in plastic tubes
Always clean your own glassware using only hot water and/or organic solvents and rinse it with milli Q grade water. Use the highest grade reagents possible. Use a set of glassware, reagents and pipettes only dedicated for MS-experiments.
Use appropriate plasticware and high quality solvents
Use Falcon tubes (for larger volumes) or Axygen low-retention 1.7 ml, 0.6 ml or 0.2 ml tubes for your experiments (1.7 ml tubes: MCT-175-L-C; 0.6 ml tubes: AX-MCT-060-L-C; 0.2 ml tubes: AX-PCR-02-L-C, distributor: genXpress). Do not touch the tubes at any stage of the sample preparation without gloves.
Filter-sterilize all your buffers and store them in fresh bottles obtained from the store instead of bottles from the wash-kitchen. Use new pipette tips from the store instead of refilled autoclaved pipette tips.
Analyse a small aliquot of your sample by SDS-PAGE
Separate a small amount (e.g. 10%) of your sample by SDS-PAGE followed by Coomassie or silver staining for quality and quantity control. Additionally, please provide all known information about your sample, such as the exact solvent composition, protein concentration and the sample preparation.
Sample preparation guidelines for protein separation by gel electrophoresis
Use pre-cast gels
If your proteins are to be separated by gel electrophoresis before MS-analysis, please use pre-cast Invitrogen/Novex Tris/glycine SDS-PAGE gels instead of casting them yourself.
Use MS-compatible staining method
Not all gel staining methods are compatible with MS-analysis. While all types of Coomassie staining are compatible, silver staining applying glutaraldehyde in the sensitizing solution is not compatible with MS. Therefore, it is necessary that the recommended silver staining procedure is used (see Protocols, silver stain protocol according to Blum). For silver staining, do not overstain the gel, as this reduces the yield of identifiable peptides from your sample. For Coomassie staining, also try to only stain your gel for the minimum time to (just) detect your protein(s) of interest. Extended staining will increase the background in subsequent MS analysis. Destain the gel thoroughly to clear the background and to enhance visibility of the band. Do NOT heat your gel in the microwave to speed up the staining!
Avoid keratin contaminations
It is essential to prevent contaminations of the gel by hair and skin particles or cloth threads, therefore carry out all staining steps in closed dishes and use filter-sterilized solutions which are dedicated to MS-experiments. Wear gloves and lab coats at each step. Be aware that any surfaces, glassware or chemicals exposed to the lab atmosphere for more than a few minutes will be contaminated with keratin as dust from the atmosphere settles. Therefore wash everything (gel tank, staining trays,..) before use and then keep it covered.
Provide a picture of the gel and all known information about the sample
Please provide a scan of the stained gel, where the bands of interest are labeled. Additionally, it is important that you provide all known facts about the nature of your sample, helping us to choose the optimal digestion and analysis strategy.
The protein bands to be analyzed by MS will be cut out in a laminar flow hood in the MS-lab by a member of the MS-unit. In case you want to have many bands analyzed (i.e. more than 10), you may be asked to cut them out yourself and to proceed with the digest in our laboratory. This also depends on the total number of samples that are waiting to be processed at that moment.