Proteins submitted in solution
Use MS-compatible buffers
If you submit protein in solution for MS analysis, they 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. Water and buffers prepared by the media kitchen can be used. 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. 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:
- Glassware previously used for buffers
- Glassware washed in a dishwasher
- Pipettes could be contaminated
- Communal lab chemicals
- 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 a set of glassware, reagents and pipettes only dedicated for MS-experiments.
Use appropriate plasticware and high quality solvents (HPLC grade)
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.
Use new pipette tips from the store instead of refilled autoclaved pipette tips.
Filter-sterilize all your buffers and store them in fresh glass bottles obtained from the store instead of bottles from the wash-kitchen. Reuse the same bottle for the same solvent when you prepare a fresh batch. Never use plastic pipettes to transfer solvents from the original bottles, instead pour the solvent into a beaker or use Pasteur or glass pipettes. Never use plastic pipettes to pipette concentrated acids (>10%) but use glass pipettes. Use the highest-grade reagents possible.
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.