Tips for correct sample handling
Tips for correct sample handling prior to mass spectrometry analysis
and other considerations
1. Watch out with keratins!
Keratins are probably the most common contamination found in protein identification analysis by mass spectrometry anywhere. Keratins are major constituents in hair and nails, and also are abundant in the many skin cells that are detached from our bodies every day. Therefore, it is very important to keep the sample away of any source of keratin. The use of lab coat and gloves are necessary during sample manipulation prior to digestion (better if gloves are powder free), but we also recommend following the next recommendations:
-Use clean reservoir for gel staining. It is better if they are glass made rather than plastic. Avoid using recipients prior used for western blot analysis (they may have traces of blocking protein, and you do not want to have this in your sample)
-Do not wear wool madden clothes unless you want sheep keratin among your results!
-Clean thorough any surface-material that will be in contact with the gel (e.g. with a clean tissue and ethanol).
2. Type of gels
A priori, a spot or band from almost any kind of polyacrylamide gel can be used for in-gel digestion and further mass spectrometry analysis. Nevertheless these are the recommendations:
- Gel type: Tris-Glycine or Tris-Tricine
- Acrylamide %: 10-12% (higher percentage can also give good results)
- Gel Thickness: 0.75 to 1 mm
Special care should be taken for in house casted gels: glasses, combs etc. should be clean up with care and stock solutions should be clean of keratins.
3. Gel staining methods
The more, the better, meaning that the more protein we start up with, the easier will be the identification. Therefore, coomassie blue stained bands are the preferred ones. Anyhow, we are very much aware that this is no always the case. Fluorescent staining methods such as Sypro Ruby or Flamingo are very sensitive and also render good results. When this is not possible, silver stain can be used. We recommend the protocol developed by Shevchenko et al. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68; 850-858. (1996).
4. How to store gels
In general, it is preferred to work with fresh gels. Anyhow, once that gels are fixed and stained, they may be stored for quite long time. They can be stored for few days in mili-q water, and for longer periods, they can rest in the fridge in 1 % acetic acid.
5. How to send-bring us the sample
The gel band, should be cut out with clean scalpel, trying to avoid bringing any excess of surrounding polyacrylamide. Once transferred to the eppendorf tube (eppendorf brand), big bands should be further cut to achieve 1 mm3 pieces. This will help the trypsin to reach to all the protein. We recommend to add mili-q water to keep the band hydrated (50 to 200 uL should be enough). Dry gel bands can be extremely easy to loose when opening the ependorff tube.
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