Policy

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

Before you bring your samples to the MS-lab, 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 Rottinger (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 form to steinmacher(at)imp.ac.at or opravil(at)imp.ac.at (Ines Steinmacher or Susanne Opravil -tel: 4298).

If you plan a larger project, where a substantial amount of time is required for the MS-analysis (i.e. more than 2 months), it is necessary that the planned project is discussed with both scientific advisors of the facility, namely Jan-Michael Peters and Jürgen Knoblich.


Sample preparation guidelines for proteins in solution

    • Use MS-compatible buffers

    Samples should not contain high amounts of salts (>100 mM), they should be free of non-volatile buffer components (e.g. phosphate buffer) and of detergents (e.g. SDS, Triton, Tween). Additionally, the sample volume should be as small as possible (opt. 20 – 100 µl) and the pH of the sample should be neutral. Use 1M TRIS buffer (pH 8.0) to neutralize your sample (you can get this buffer in the Media kitchen). In case you are planning a quantification use 1M TEAB instead of TRIS (please get this buffer from our MS-Lab). We also provide a protocol for protein immunoprecipitation, which is compatible with MS.

    • Use appropriate plasticware and high quality solvents

    Use Falcon tubes (for larger volumes) or Axygen low-retention 1.7ml, 0.6ml or 0.2ml tubes for your experiments (1.7ml tubes: MCT-175-L-C; 0.6ml tubes: AX-MCT-060-L-C; 0.2ml 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

    Mark Petronczki producing his first Cell paper

     

    • 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 (pre-cast gels).

     

    • Use MS-compatible staining method

    Not all gel staining methods are compatible with MS-analysis. While Coomassie staining techniques 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.

     

    • 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.

    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, this helps us 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 analysed (i.e. more than 10), you may be asked to cut them out yourself and to proceed with the digest in the MS-lab. This also depends on the total number of samples that are waiting to be processed at that moment.

     

    • Proteolytic digest and analysis

    Depending on the scientific question to be addressed in the specific case, the samples can be digested with different proteolytic enzymes (see protocols in gel digest), eventually further processed and finally analysed by an appropriate mass spectrometer.