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 MS experiment with either Karl Mechtler or another facilty member before you generate the samples.
Before you submit your samples to the protein chemistry facility, you must contact Karl Mechtler (karl.mechtler(at)imp.ac.at, VBC6 ground floor, room number E71, tel: 4280) to discuss your analytic question. In case Karl Mechtler is not available, please contact Elisabeth Roitinger (elisabeth.roitinger(at)imba.oeaw.ac.at, VBC6 ground floor, room number E78, tel: 4278). For the actual submission of your samples please use the MS Submission Portal.
To avoid any unnecessary contact with our facility members please use the fridge/freezer which is set up at IMBA Plaza floor next to the copy machine to submit your samples.
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, ETDA, Glycerol), 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 case, TMT-based quantification of your proteins is planned, do NOT use TRIS buffer but TEAB instead (please get this buffer from our laboratory). 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 maximum recovery tubes for your MS-experiments, which you can get in our facility on a limited scale(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, Szabo-Scandic). 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 proteins bound to beads
Please keep the volume of beads as small as possible. You can determine the optimal amount of beads and bead to extract ratio in a western blot before submitting your samples. Following the immunoprecipitation, transfer the beads to a new tube (preferentially Axygen low binding tubes – available in the Protein Chemistry Facility), wash your beads several times with your preferred buffer, which has to be free of detergents, free of protease inhibitors and, if possible, free of glycerol and EDTA (e.g. 6 times with 20mM Tris pH 7.5 to 8.5, 75 mM NaCl).
Take a small aliquot (5-10%) of the beads during your last wash step and analyse it by both, silver staining and Western blot. For the silver stained gel please also include a known amount of a protein ladder or different amounts of a BSA standard. For the Western blot please include your input lysate and a similar amount of the supernatant after the IP to be able to evaluate the success of the IP. In the optimal case, the bait protein shows a reduced signal in the supernatant but is not depleted from the lysate (see picture). After the last wash step remove the supernatant and submit the beads either frozen or at +5°C.
We can only accept your submission, if it contains an appropriately labelled picture of the silver-stained gel and Western blot.
In case you resubmit a similar experiment, we recommend controlling it again as described above, but we will accept the submission also with one of both pictures. In this case please make a reference to the corresponding previous submission.
Sample preparation guidelines for proteins separated 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/NuPAGE Bis-Tris 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.
Sample preparation guidelines for whole cell lysates or pellets
For MS sample preparation of cell pellets derived from cultured cells (for TMT, PRM experiments) we will from now on offer a robust and fast protocol which is provided as a kit by the company PreOmics. The protocol is easy to perform and can be done in your lab and will produce ready-to-measure peptides, which can be submitted to our facility. For detail questions please contact Michael Schutzbier or Karel Stejskal.
- The protocol is adjusted to protein amounts of 10 to 100 microgram (5x10e5 HeLa cells appr. yield 150 microgram). We recommend using the Micro BCA Protein Assay to measure protein content after cell lysis.
- We will provide aliquots of the kit needed per reaction in our sample submission fridge next to the copy machine near room nr 5.44 at IMBA Plaza.
- We will have to pass on the costs of the kit (28 Euro per reaction) to your group.
- With this measure we want to speed up the sample processing and increase the throughput of samples in our facility.
- Please contact Karl Mechtler, if you want to submit a complex sample and use the kit.
- We provide the protocol with some adjustments on our homepage at cores.imp.ac.at/protein-chemistry/protocols/ (this section is only accessible within the IMP/IMBA/GMI network).