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SWISS-2DPAGE

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Amino acid composition

 There has been a recent revival of interest in the use of AA composition for the identification of proteins from 2-D gels. This technique uses the idiosyncratic AA composition profile of a protein in order to identify it by comparison with theoretical AA compositions of proteins in databases. For identification of proteins from 2-D gels, we match the AA composition in conjunction with estimated protein pI and Mw. Protein identification by compositional analysis is best used when there is sufficient sample available for micropreparative 2-D PAGE, as a minimum of 250 ng of protein per spot of interest is required. As the approach is rapid, inexpensive, and produces easily interpreted data, it is suited for the screening of large numbers of proteins from 2-D reference maps. We can analyze 20 PVDF-bound proteins per day on a single AA analysis station. Rapid methods for the AA analysis of PVDF-bound proteins are presented below. These methods have been optimized for use with samples prepared by micropreparative 2-D and blotted to PVDF in a glycine-free buffer. To control for variation in AA analysis results, we always analyze samples in batches. Each batch comprises a calibration protein (PVDF-bound bovine serum albumin) and 12 samples. The batch is hydrolyzed together, AAs are extracted using common solutions, and the AA analysis of each batch is carried out sequentially on the analysis instrument. After AA analysis, the analysis quality of the calibration protein is checked as a benchmark, and its analysis data is used to adjust that from unknown spots during protein identification by database searching.

Hydrolysis of PVDF-bound proteins

Vapor-phase protein hydrolysis requires that you have a hydrolysis vessel, and vessel holder. This design is recommended as other vessels (even some which are commercially available) are not able to withstand repeated heating to 155oC in the presence of HCl vapor. Access to a vacuum source and fume hood are also required. At all times, contamination of samples with dust, skin, hair and breath must be avoided. It is advisable to wear powder-free latex gloves and work in a clean environment.

Vapor-phase hydrolysis of protein spots from 2-D gels for amino acid analysis

  1. Cut spots of interest from PVDF membranes with a scalpel or cold punch, on a minimum of PVDF.
  2. Place each spot into a 700 µl clear glass autosampler vial. If possible, use vials that can later be used directly on your AA analysis autosampler. If spots are very feint and/or small, multiples of identical spots from different gels can be placed into a single vial. Use a diamond pen or glass engraving tool to label vials.
  3. Place 400 µl of 5.7 N HCl (BDH Aristar Grade) and a crystal of AR-grade phenol (approximately 3 mm long by 0.5 mm wide) into the bottom of the hydrolysis vessel. Note that these reagents do not go into the autosampler vials containing the samples.
  4. Using a pair of stainless steel tweezers, place up to 13 autosampler vials into the hydrolysis vessel so they are upright. Include one vial containing a calibration protein.
  5. Assemble the hydrolysis vessel tightly. Evacuate the vessel for 10 sec (the acid should boil), and then flush with argon. Repeat the evacuation / flush steps. Finally evacuate, close the tap on the vessel, and place the vessel into the preheated vessel holder in the 155o C oven for 1 h.
  6. After heating, remove the vessel from the oven and transfer it to a fume hood. Release the acid vapor from the vessel by opening the tap, dismantle the vessel, and remove the autosampler vials with tweezers. These steps should be done immediately to prevent condensation of acid forming on the samples. CAUTION: Vessels must be opened in a fume hood, as hot HCl vapor is very dangerous! Eye and hand protection must be worn!
  7. Dry the autosampler vials containing the PVDF membranes under vacuum for 10 min (Savant Speedvac) to remove residual HCl vapor.

Post-hydrolysis extraction of amino acids from PVDF

 After hydrolysis of PVDF-bound proteins, AAs are extracted from the membranes in preparation for AA analysis. To minimize sample manipulation, samples are kept in the same vial for the entire extraction procedure. A sonicating water bath is required. Note that step (e) of this protocol resuspends the extracted AAs in 250 mM sodium borate buffer pH 8.8 in preparation for AA analysis using 9-fluorenylmethyl chloroformate (Fmoc). This buffer may, however, not be suitable if AA analysis is to be done using other derivatisation chemistries.

Post-hydrolysis extraction of amino acids from PVDF

  1. Add 180 µl of fresh extraction solution (60% v/v acetonitrile in 0.01% v/v trifluoroacetic acid) to each autosampler vial containing a PVDF spot. Make sure the PVDF is submerged in the solution.
  2. Cover each vial with parafilm, position in a polystyrene floater, and place the floater into a sonicating water bath (21o C) for 10 min.
  3. Discard the parafilm covers of the vials. Remove the PVDF membrane from each autosampler vial with a hypodermic needle and discard. Keep the hydrolysate in the autosampler vial. Vial to vial sample carryover should be avoided by either using only one needle per vial or rinsing the needle between vials in fresh extraction solution.
  4. Place the autosampler vials containing protein hydrolysates in a vacuum dryer (Savant Speedvac) and evaporate to dryness.
  5. Add 10 to 20 µl 250 mM sodium borate buffer pH 8.8 to each autosampler vial, mixing carefully to ensure all AAs are dissolved. The samples are now ready for AA analysis.

Derivatisation and chromatography

 The AA analysis of protein hydrolysates is achieved using a modified Fmoc precolumn derivatisation method which is carried out at room temperature, produces monosubstitued forms of His and Tyr, and does not require the removal of excess Fmoc before chromatography. Derivatisation of AAs should be carried out in the same glass vial that was used for hydrolysis and extraction. We derivatise AA standards (Sigma # AA-S-18) to check derivatisation and chromatography efficiency and to allow quantitation of samples. Note that the Fmoc-amino acid derivatives are stable for 24 h, allowing many samples to be prepared in advance and loaded onto an autosampler for injection. Alternatively, the derivatisation can be done by any autosampler which has minimal vial to vial sample carryover and can accurately manipulate 10 µl volumes. We use a GBC Aminomate Amino Acid Analyzer (GBC Scientific Instruments, Dandenong, Vic., Australia) for this purpose.

Derivatisation of amino acids with 9-fluorenylmethyl chloroformate

  1. Ensure that all reagents and samples are at room temperature.
  2. Starting with 10 µl of hydrolysate, add 10 µl of Fmoc reagent and mix thoroughly by pipetting up and down. Wait for 90 sec.
  3. Add 10 µl of cleavage reagent and mix thoroughly by pipetting. Wait for 3 min 30 sec.
  4. Add 10 µl of quenching reagent and mix thoroughly by pipetting.
  5. The mixture can now be injected directly into the HPLC system, or sealed and stored for up to 24 h.

Amino acid composition was then matched, in conjunction with the estimated pI and molecular weight of the protein with the corespondent protein in Swiss-Prot database. The matching algorithm calculates the least squared distance between the theoretical and measured amino percentage for each protein (see AACompIdent tool for details). A score was obtained and a typical pattern was defined for assignments.


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