| | Proteins may be identified by several related methods and platforms. The steps involved are similar in purpose regardless of the application employed. The basic steps are protein separation, chemical and/or proteolytic digestion of any protein or peptide that is larger than 4 kDa, mass-to-charge ratio measurements on the fragments produced in the digestion by mass spectrometry (MS) and database matching of fragment masses. Protein separation is generally achieved through a series of procedures. Initially a subset of cellular protein is prepared which could include soluble fraction, membrane fraction, mitochondrial preparations, nuclear preparations etc. For proteins in solution such as a cellular fraction, serum, CSF, urine etc., additional separations should be performed based on some physical property such as isoelectric point (pI), molecular weight, polarity, immunoaffinity, HPLC, MDLC, etc. Finally, some combination of separation strategies must yield a protein fraction with a complexity of 1-3 protein species which may be able to be identified by PMF or a peptide which can be analyzed by MS/MS for amino acid content or sequence.
1D gel preparation for trypsin digestion
A 1D PAGE gel of appropriate % acrylamide (based on molecular weight range of interest) is electrophoresed. The gel is stained using an MS-friendly stain such as BioRad Bio-Safe Coommassie Blue with a 2 h destain in DI water, or Invitrogen Silver Quest Kit stain when more sensitivity is required. Note that fixatives (e.g., glutaraldehyde, formaldehyde, high concentrations and long presence of methanol) MUST be avoided. The band of interest is then picked from the gel using a sterile, disposable gel-plugging tip 1mm in diameter. The gel band can be stored in up to 1% aqueous acetic acid at 4°C for up to 48 h prior to trypsin digestion. Alternatively, the gel plugs may be incubated with 1:1 (v/v) MeOH: 50 mM NH4HCO3 and dehydrated in acetonitrile, then dried in a SpeedVac for storage at -80°C for up to 3 months.
2D gel preparation for tryptic digestion
The GPCL will perform traditional 2D and difference (DiGE) gel electrophoresis upon request. The GPCL staff will train and assist in data analysis at an hourly rate beyond an initial 2 h training at no charge. Once a list of protein spots to be picked has been chosen, the gel is re-imaged, gel plugs are excised from the gel and, depending on sample number and picking method, placed into tubes or microtiter plates.
For DiGE the gel plugs may only be excised using an automated picker into microtiter. For traditional 2D gels, the gel plugs may be excised manually or with an automated picker. If traditional methods are used, the gel must be stained with an MS-friendly stain (see above).
An investigator may also provide gel plugs, which the Core will process directly.
Gel and Gel plug storage
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Gel pieces for trypsin digestion and protein ID should be processed as soon as possible after the gel has been electrophoresed. A gel or gel plug may be stored up to 48 h in 1% acetic acid prior to digestion. Beyond 48 h, some loss of protein coverage will be observed and may reduce the confidence in MS-based protein ID. Alternatively, the gel plugs may be prepared for long term storage as detailed above. Up to two gel plugs from the same protein spot/band should be incubated in 1:1 (v/v) MeOH: 50mM NH4HCO3 twice (30 min each incubation) at room temperature. For a 1 mm diameter plug cut from a 1 mm thickness PAGE gel, 100 µl should be used. Buffers are then carefully removed, the plug is dehydrated in 50 µl of acetonitrile for 15 min, then placed in a SpeedVac at room temperature until dry (about 45 min). Following dehydration, the gel plugs may be stored at -80°C for up to 3 months; the gel plug should have an opaque/white appearance.
Peptide Mass Fingerprinting
Trypsin, the quantity of which is determined based on the protein load in the sample, is added to the gel plug. This mixture is incubated at 42°C for 2 h, then extracted with in 1:1 acetonitrile:H2O containing 0.3% aqueous trifluoroacetic acid (TFA). The supernatant is dried in a SpeedVac and resuspended with in the same solvent, mixed with MALDI matrix and immediately spotted onto the MALDI target for MS and MS/MS analysis. The mass spectra for each tryptic digest will be measured. The analysis will be performed with the ABI 4700 instrument in reflectron mode. The resolution of the instrument, which has a 3.75 m ion flight path, at 2.5 kDa is > 12,000, and mass accuracies of +/- 5 ppm with internal standardization and +/- 25 ppm with external standardization are routinely achieved. This allows for ID of proteins within gel spots or bands containing 1-4 proteins.
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