The facility operates three Hitachi (L-8800, L-8800A & L-8900) amino acid analyzers. The L-8900 Hitachi analyzer utilizes a lithium citrate buffer system and is optimized for physiological samples and is capable of quantitating more than 40 different amino acids utilizing a lithium citrate buffer system. The L-8800 Hitachi analyzer utilizes a sodium citrate buffer system and is optimized for hydrolyzed proteins/peptides. All analyzers use ion-exchange chromatography to separate amino acids followed by a "post-column" ninhydrin reaction detection system. The Na system can quantify individual amino acids down to the pmole level (~100 pm). Our standard hydrolysis procedure employs 6N HCl for 24 hrs at 110 C. Alternate procedures are available for the determination of tryptophan, cysteine and methionine. Physiological samples are usually acidified with sulfosalicyclic acid to remove any intact proteins prior to analysis, but sample composition determines the optimal preparation protocol.
The Molecular Structure Facility's Hitachi L-8800 Na citrate-based amino acid analyzer is routinely used to establish the quality/quantity of synthetic peptides, to determine the concentration of protein and to calibrate protein-dye assays.
The amount of protein/peptide required for quantification is:
|preferred||25. ugram||10. nmole|
|minimum||5. ugram||2. nmole|
|detection limit||1. ugram||0.4 nmole|
The Li citrate-based analyzers are routinely used to determine the free amino acid concentrations in all types of physiological samples (plasma, fermentation media, tissue extracts, etc.). Since these machines are capable of resolving more than 40 different amino acids, they are especially useful in the quantification of usual or modified amino acids. Physiological samples are usually acidified with sulfosalicyclic acid to precipitate any intact protein prior to analysis.
While these analyzers are fairly tolerant of sample impurities, there are some common contaminants (urea, detergents, high concentrations of buffer/salts, etc.) which are incompatible with ion-exchange chromatography. Samples in small volumes (~200 ul) and/or low concentrations (~50 mM) of simple buffers are generally suitable for analysis. Acrylamide contaminants must be removed from samples purified on PAGE by electroblotting or electroelution. Most biological buffers and detergents, even at low concentrations, will interfere with AAA. We strongly encourage consultation with the MSF staff prior to submitting any sample.
Cysteine, Methionine and Tryptophan
Cysteine (& cystine), methionine and tryptophan are destroyed during hydrolysis with 6N HCl. Cys and Met can be determined by oxidation with performic acid, yielding the acid stable forms cysteic acid and methionine sulfone, prior to the standard acid hydrolysis. The conversion of met is quantitative while that of cysteine is >90%. Tryptophan can quantitated but requires consultation with the MSF staff.
All assays based upon a protein-dye binding reaction (Bradford, BCA, Lowry, etc.) are unreliable for determining the concentration of proteins. The values generated can differ greatly from the true protein concentration. These dye assays should be calibrated for each protein using AAA. This standardization will result in a much more reliable assay for determining the relative concentration of a protein. However, determination of the absolute concentration of the protein will still require AAA.
AAA Data Analysis
A data file comprised of a series of absorbance values vs. time points is generated during each chromatographic run. This file is stored on hard disk and is used by the software to generate a report identifying and quantifying each amino acid present.
The "Amino Acid Analysis Run Sheet" lists the run numbers, the corresponding sample ID's and the sample volumes. In most cases, the sample will have been dissolved in Na citrate buffer containing 40 nmoles/ml Nle or in Li citrate buffer containing 100 nmoles/ml AEcys. The standard injection volume is 50.0 ul. Nle and AEcys are internal standards used to correct for any variation in the operating conditions of the analyser over time.
The Beckman System Gold software identifies any peak which falls within predetermined limits of known amino acid retention times and prints this data in the column labelled "Component Name". The area of each identified peak is multiplied by its responce factor and this result is printed in the column labelled "Concentration". The responce factors are calculated from multiple injections of a known standard run concurrently with the samples.
Each amino acid's identification and quantitation is double-checked and underlined. Values for Pro (Asn and Hypro when present) are transferred from the 440nm printout. As the computer software can sometimes be "fooled", there may be handwritten corrections on the printouts. All reports are reviewed by two staff members prior to release.