SPECIAL FEATURE

The following article is the second response to a solicitation by the Amino Acid Analysis and Education research committees for information on the methodology used for analyzing the ABRF92AAA sample. We had chosen to send the solicitation to the "best" sites of the 1992 study with "best" defined as having both the lowest percent error in analysis in each specific category of instrumentation (such as pre-column PITC, pre-column fluorescence, post-column fluorescence, etc.)and having also done an analysis for Cys and Trp, which amino acids were the focus of the 1992 study. Anonymity was preserved and participation was voluntary as described with the first article which appeared in the June 1993 edition of this newsletter.

The Amino Acid Analysis Research committee would like to caution readers to consider the specific purpose of an analysis when deciding on a methodology. The methods for Cys and Trp estimation presented here provide good examples for the semiquantitative estimation of these amino acids, as some users may require. On the other hand, the 1992 study has pointed out various workable methodologies for instances when good quantitation is required and readers are referred to that study for further details (Strydom, D.J., et al in "Techniques in Protein Chemistry IV", Angeletti, R.H., Ed., Academic Press, San Diego, 1993. pp 279-288). Quantitative analyses of both Cys and Trp require adequate chromatographic separation as well as reproducible and quantitative derivatization and release during hydrolysis.

Amino Acid Analysis of ABRF92-AAA Sample

Ragna Sack and Peter E. Hunziker, Biochemisches Institut der Universitat Zurich, Winterthurerstr.190, CH-8057 Zurich, Switzerland

During the history of amino acid analysis at our establishment a variety of instruments and methods have been employed. Starting with automatic analyzers from Beckman more than 22 years ago, the institute bought two Durrum D500 machines which were run successfully for more than 10 years. These employed ion-exchange chromatography for the separation of free amino acids followed by their reaction with ninhydrin and photometric detection of the products. Typical run-times for protein hydrolysates were about two hours per analysis and 5 to 10 nmol of sample were needed. With the increasing demand for higher sensitivity and shorter run-times we tried to establish pre-column derivatization/reversed-phase HPLC methodologies in our facility. Since at that time only a small number of suitable instruments were starting to become commercially available, we built up an analyzer on the basis of common HPLC equipment using the separation and fluorometric detection of dansylated amino acids (De Jong, C., Hughes, G.J., van Wieringen, E. and Wilson, K.J. (1982) J. Chromatogr. 241, 345-359). The sensitivity was very high but most researchers at our institute were not yet prepared to handle pmol amounts of material. After a short return to the well proven ion-exchange chromatography and after gaining experience with low-level sample preparation, amino acid analysis by precolumn derivatization/HPLC separation was finally established. Currently two instruments are used in our facility: an AminoQuant from Hewlett-Packard with fluorometric detection of the o-phthalaldehyde (OPA)/9-fluorenylmethylchloroformate(FmocCl) derivatives of primary/secondary amines and a Model 420A/H analyzer from Applied Biosystems (ABI) with photometric detection of phenylthiocarbamyl(PTC) derivatives of amino acids.

Except for the Model 420A/H analyzer, which is equipped with an on-line hydrolyzer, our methods for the manual hydrolysis of proteins and peptides have changed only slightly over the years. We still hydrolyse the samples at 110deg.C for 22 -24 hours, but the hydrolysis in liquid 6N HCl under vacuum was replaced by vapor-phase hydrolysis under an argon atmosphere.

Methods:

The ABRF92-AAA sample was hydrolysed manually and analyzed on the AminoQuant using our standard protocol:

Column: AminoQuant column (79916AA-572) with guard column (79916KT- 110) from Hewlett-Packard.

Buffer Stock Solutions: Buffer A (5x):

100 mM sodium acetate, 0.5 mM EDTA (disodium salt),

0.09% (v/v) triethylamine; pH 7.2 adjusted with 0.25 M

acetic acid

Buffer B (5x):

100 mM sodium acetate; pH 7.2 adjusted with 0.25 M acetic

Running buffers: Buffer A:

100 ml stock solution, 400 ml H2O, 2 ml tetrahydrofuran

Buffer B:

50 ml stock solution, 100 ml methanol, 100 ml acetonitrile

Both running buffers are freshly prepared every day.

Flow-rate and gradient:

According to the AminoQuant 11 manual: 0 - 60% buffer B in 17 min (0.45 ml/min); 60 - 100% buffer B in 1 min; 100% buffer B for 6 min (0.80 ml/min); 100 - 0% buffer B in I min (0.45 ml/min)

Sample injection:

The following autosampler program is used: Withdraw 5 ul of buffer (0.4 N borate, pH 10.2; Hewlett-Packard #5061-3339), 1 ul OPA solution (Hewlett-Packard#5061-3335) and 1 ul of the amino acid standard solution or 1-2 ul of the sample solution. Mix 6 times. Withdraw 1 ul Fmoc-CI solution (Hewlett-Packard #5061-3337). Mix 3 times and inject.

Amino acid standards and calibration:

Stock standard solutions (100,25 and 10 pmol/ul) are purchased from Hewlett-Packard. Solutions for calibration are prepared as follows: 900 u1 of each standard stock solution are diluted with 100 ul of H2O containing I nmol/ul each of norvaline and sarcosine (internal standards for primary and secondary amines, respectively). Aliquots of 100 ul are transferred into sample tubes, closed with crimp caps and stored at 4deg.C until use. For each batch routine the system is calibrated with all three standards (90, 22.5 and 9 pmol of amino acids) using the internal standard method (100 pmol of both internal standards). Trp and CySO3 (each with 90 pmol/ul in 50 mM HCl containing 100 pmol/ul of both internal standard) are calibrated as required with one standard only.

Sample hydrolysis:

Sample tubes (Chromacol Gold, 03-CVG, 0.3 ml crimp top vials) are heated at 500deg.C for 1.5 hours in a beaker covered with aluminum foil. A maximum of 200111 Of sample dissolved in a volatile solvent (prepared and desalted by our customers using any appropriate method) are transferred into a sample tube, diluted with 10-20 ul of isopropanol and completely dried in a Speed-Vac (Savant). Six tubes are placed in a screw-cap vial (Pyrex, France, 22) which has been modified as shown schematically in Fig. lA. (16k) To keep the sample tubes in an upright position we made teflon holders that are placed at the bottom of the vials (Fig. I B).

Approximately l ml of argon purged (10 min at room temperature) 6 N HCl (ABI) and a few crystals of solid phenol are added around the sample tubes. The HCl solution is again argon-purged for approximately 5 min. The sample tubes are purged using a pasteur pipette that is slowly moved from the bottom to the top of each tube. After displacing the air above the HCl solution with argon for approximately 10 min, the screw-cap vial is closed and placed in an oven. After 22 hours at 110deg.C the vial is opened immediately to prevent any HCl vapor condensing back into the sample tubes. These are then removed with forceps and placed in a Speed-Vac. The dried samples are dissolved in at least 10 ul 50 mM HCl containing 50 pmol/ill each of norvaline and sarcosine, sealed with a crimp-cap and placed in the autosampler of the analyzer.

Trp analysis (performed on request only):

Usually, 10 ul of distilled thioglycolic acid (TGA) is added to the phenolic HCl solution and the samples are hydrolyzed as described above. In order to obtain more quantitative Trp recovery, the ABRF sample was hydrolyzed at 160deg.C for 30 min.

Cys analysis (performed on request only):

Cys is converted to CySO3 by performic acid oxidation. Performic acid is prepared by incubating 950 ul formic acid and 50 ul Perhydrol (30% H2O2) for 2 - 3 hours at room temperature. The dried samples are dissolved in 10 ul formic acid and 20 ul performic acid are added. After incubation for 2 hours on ice, 20 ul of H2O are added, the samples are dried in a Speed-Vac and subsequently hydrolyzed. Since the OPA derivatives of CySO3 and Asp coelute on the separation system, the same sample is analyzed twice, once with and once without oxidation. The CySO3 content is then calculated by subtracting the integrated Asp peak area of the non-oxidized sample from that of the oxidized.

Results:

Table I summarizes the analysis of the ABRF92-AAA sample as submitted to the ABRF Amino Acid Analysis Subcommittee. (24k) With a mean error of approximately 10%, the result was within the limit we usually expect from a single amino acid analysis. Unfortunately, the CySO3 standard used with the original data (Table 1, column 3 and 4) was not normalized to the internal standard. After normalization to 100 pmol norvaline, the number of Cys residues was closer to the theoretical value (Table 1, column 5 and 6).An actual chromatogram of an ABRF92-AAA analysis is shown in Fig. 2.(16k)

Six percent (3 ug) of the sample was hydrolyzed for 22 hours in the presence of phenol and TGA. Under these conditions the yield of Trp is low but usually meets the requirements of our customers. In order to improve the Trp data for the ABRF test sample we used shorter hydrolysis times at a higher temperature. As shown in Fig. 3 (16k) , hydrolysis for 30 min at 160 deg.C resulted in a significantly higher Trp recovery.

In Fig. 4 (16k) (see above) the calculated sum of the Asp and CySO3 areas are compared with the corresponding measured values for various mixtures of the two amino acids. At higher Asp/CySO3 ratios the measured values are usually higher than the sum of the areas of the individual amino acids. However, the largest deviation is only 3.7% (approximately 10 area units) and corresponds to an absolute difference of 4 pmol in a total of 120 pmol of amino acids.

With the instrumentation and the methods described we usually obtain reliable amino acid compositions. Together with the ABI Model 420 A/H on which performic acid oxidations and/or hydrolyses are performed automatically, the equipment of our laboratory fulfills our current needs. Nevertheless, we occasionally miss the advantages of ion exchange chromatography and post-column derivatization. Therefore the purchase of the corresponding equipment will be considered in the future when replacements become due.

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