Location
- Research Support Building (CACTUS)
- Rúa de Constantino Candeira, 1. Campus Vida , 15782Santiago de Compostela
- Phones
- 881 816 242
Proteins are digested with an enzyme, usually trypsin (protease that specifically breaks up arginine and lysine residues if they are not bound to a proline), for subsequent mass spectrometric analysis of the peptides obtained.
- In-gel digestion: the Millipore In-Gel DigestZp kit is used to digest proteins resolved via 1D and 2D electrophoresis and gels stained with comassie, silver or fluorescence.
- In-solution digestion: tryptic in-solution digestion of samples is performed.
- Proteins in gel. The gel bands (1-4 mm) will be sent in an eppendorf tube in milli-Q water, with the name of the samples indicated. The bands will be cut out of the gel strictly following the contour of the gel in order not to increase the proportion of acrylamide in the sample. The recommendations indicated in the sample preparation section will be followed. The greater the amount of protein present on the spot, the easier it will be to identify it, as only a part of it will be recovered for analysis in the mass spectrometer. Good results can be obtained with 100 fmoles of starting protein with the Millipore kit.
- Proteins in solution. Proteins (1-10µg) will be sent in an eppendorf tube, with the sample name indicated. The recommendations indicated in the sample preparation section will be followed. The samples will be delivered to the Mass and Proteomics Unit, together with the duly completed application form and an image of the gel from which the samples are to be analysed (if the proteins were made in gel).
- Storage of the gels: Gels may be stored wet for a few weeks in 1 % acetic acid at 4 °C. Strips cut out of the gel can be stored frozen in water or 1 % acetic acid. Dried gels can still be valid, even after several years of storage, provided that their surfaces were not contaminated with fingerprints or dust, on which keratins are present. However, it is better to work with wet gels free of plastic or cellulose coatings.
- Compatible gels:
- Types of gels: Any type of polyacrylamide gel can be valid; what is essential is that its thickness is between 1 and 1.5 mm and that the percentage of acrylamide (which can be used in this kit) is 4-20 %. The size of the gel piece would be 1-3 mm in diameter for 1D gels and 1-2 mm in diameter for 2D gels. Special care should be taken not to introduce foreign substances into the gel that could interfere with the mass spectrometric analysis. Therefore, it is advisable to prolong the polymerisation overnight to avoid the presence of unpolymerised acrylamide residues.
- Staining methods: Comassie, silver (avoid using Glutaraldehyde), fluorescence.
- Reagents compatible with mass spectrometry:
- Solvents: Use reagents of HPLC purity grade and milli-Q water.
- Non-interfering: TFA, formic acid, Beta-Mercaptoethanol, DTT, volatile organic solvents, HCL, NH4OH, acetic acid.
- Tolerable at lower concentration than indicated:
- < 2M: Urea Guanidine
- < 50mM: HEPES, MOPS, Tris, NH4O, Octyl glucoside acid.
- < 10mM: Phosphates
- <1 %(V/V): Glycerol, n-octyl-b-glucopyranoside, LDAO.
- <0.1 %(V/V): Sodium azide, PEG2000, Triton X-100, RTX-100, NP-40, Tween, Zwitte.
- < 0.01 %(V/V): SDS, CHAPS.
- To avoid: DMSO, DMF, NaOAc, NaCL.
- Handling of specimens and gels: surfaces and material must be perfectly clean. A scalpel and forceps, or a conveniently trimmed disposable tip, can be used for excision, always taking care that this material is completely clean.
- Contamination from keratins: It is essential to work as cleanly as possible, as the incorporation of contaminating proteins makes it difficult or impossible to identify the protein of interest. The most frequent contaminants are bovine serum albumin and human keratins, the latter being abundant in dust, fingerprints and hair. For this reason, special emphasis should be placed on the cleanliness of the crystals used in electrophoresis and fresh dye solutions should be used. The gel should never be touched with fingers, scalpels or tweezers, and any material, which is used to cut out stains, should be cleaned.
Mass Spectrometry and Proteomics Unit
- Research Support Building (CACTUS)
- Rúa de Constantino Candeira, 1. Campus Vida , 15782Santiago de Compostela
- 881 816 242