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 proteinsVapor-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
Post-hydrolysis extraction of amino acids from PVDFAfter 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
Derivatisation and chromatographyThe 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