Amino acid analysis, in and of itself, is still one of the foremost measurements of the biological sciences, and is also a desirable (some would say necessary) prerequisite for other techniques such as Edman degradation, mass spectrometry, and other sequencing approaches. The Amino Acid Analysis Research Committee has noted that during the past several years of collaborative trials, the average error among participants has been 10 - 12%. Based on this observation, the Committee has described amino acid analysis as "deceptively difficult" (1). Therefore, the Committee sponsored this workshop because it seemed to be an appropriate forum to review practical, fundamental procedures necessary to obtain high quality analyses. It was not the objective of the workshop to cover exhaustively all possible approaches to AAA but rather to review the major techniques employed by ABRF sites and to highlight the most likely sources of error in routine practice. Umit Yuksel (University of North Texas Health Science Center at Fort Worth) discussed hydrolysis, Lowell Ericsson (University of Washington) reviewed post-column technology, Tom Andersen (Albany Medical College) reviewed pre-column technology, concentrating on PITC, and Izydor Apostal (Sommatogen, Inc.) discussed the newer AccQ-Tag approach. Finally, Jay Fox (University of Virginia) reviewed data handling.
Careful hydrolysis of protein or peptide samples is one of the crucial steps for successful amino acid analysis. This was demonstrated clearly by the results of the ABRF- 94AAA collaborative study (1) in which a sample which was pre-hydrolyzed by the AAA Committee was analyzed with 6.5 +/- 4.0 % error while a similar sample which was hydrolyzed individually by each site resulted in 10.9 +/- 3.7 % error. In order to obtain good analyses, it is essential to be very clean and to use the highest quality reagents available; many workers prefer to use pre-made (and pre-tested) solvents. Even fingerprints can cause contamination, and can be avoided by using gloves, but one must be careful not to allow glove powders such as cornstarch to generate an alternate contamination. Fingerprints and dust can be eliminated by starting with clean hydrolysis tubes, which should be washed with acid prior to use. Commonly used procedures are to boil the tubes for 1 h in 1 N HCl, or to soak at room temperature in concentrated HNO3, or concentrated HCl/concentrated HNO3 (1:1 v/v), always followed by extensive washing with high purity water. Tubes are usually dried in an oven overnight and kept under cover until use. Pyrolyzing the glassware at several hundred degrees is also useful. Some glass tends to bind Asp and Glu, so that attention to the manufacturer is sometimes necessary. Other common sources of contamination include carryover from previous samples, and washing of the injector port and syringe between samples is a must.
While base and enzymatic hydrolysis have special utility, acid hydrolysis (6 N HCl) is overwhelmingly the most common technique. Liquid phase hydrolysis, based on the work of Stein and Moore (2), is considered the standard. Acid must be of the highest purity and is conveniently obtained in 1 mL ampoules from Pierce. Samples are hydrolyzed under vacuum (or inert atmosphere to prevent oxidation) at 110 C for 24 hours. For vapor phase hydrolysis, one or more vials containing the sample(s) are placed in another larger vessel. HCl is added to the bottom of the outer vial which is then evacuated, sealed and heated. Since only the acid vapor reaches the sample, contamination from acid is minimized and the procedure is advantageous for micro- analysis. Care must be taken to ensure that condensation of the acid into the tubes does not occur. Drawbacks to acid hydrolysis include complete conversion of Asn and Gln to Asp and Glu, respectively, complete loss of Trp, partial destruction of Ser and Thr, and incomplete hydrolysis between beta-branched amino acids (Val-Val, Ile-Ile). Remedies for many of these have been described. For Asn and Gln, the sample may be treated with bis(1,1- trifluroacetoxy)iodobenzene, thereby obtaining diaminopropionic and diaminobutyric acid, respectively (3). Extending the hydrolysis time to 48 and 72 hours provides a means to extrapolate back to zero time to obtain the actual values for Ser, Val, Thr, and Ile. Additions of fresh preparations of 0.1% (w/v) phenol and 1% 2-mercaptoethanol to 6 N HCl prevents halogenation of tyrosine and oxidation of methionine to methionine sulfoxide. Additives to preserve tryptophan include dodecanethiol (4) and thioglycolic acid (5), among others (6).
Phosphorylated residues can be analyzed as well, although phosphorylated Ser, Thr, and Tyr have varying degrees of stability. Highest recoveries of PSer and PTyr are produced with hydrolysis times of 60 min or less; for PThr, hydrolysis times of 2 h or longer were better (7). In all cases, recoveries were only about 30-60%. For PTyr, better recoveries can be obtained with alkaline hydrolysis (5 M KOH at 155oC for 30-60 min or 110 C for 18 h). Near quantitative recovery of PSer can be obtained by conversion to a more stable derivative. Phosphate can be removed by beta-elimination followed by reaction of the resultant dehydroalanine with ethylmercaptan to form the stable S- alkyl cysteine. O-glycosylated Ser will produce the same derivative.
Amino acid analysis of PVDF-bound proteins is relatively straightforward except that glycine and TRIS are common contaminants. This is not a major problem if the samples are washed first with the destaining solution, then thoroughly rinsed with HPLC-grade water. As with other procedures, it is important to run system and sample blanks to account for amino acids and other contaminants from the membranes. Direct hydrolysis of the protein on the membrane is feasible. PVDF strips (2x10 mm) containing Coomassie- stained protein are placed into 2 mL glass ampoules, 200 uL 6 N HCl is added, and the ampoule is sealed and heated at 110 C for 24 h. The sample is dried under vacuum, reconstituted with 200 uL water, spun in an Eppendorf tube and the membrane removed with tweezers.
Ion exchange chromatography for the analysis of amino acids was first automated by William Stein and Stanford Moore (2) at Rockefeller University some 30 years ago. There have been many improvements over the years, especially in the first 20 years following their contributions. There have been relatively few methodological advancements in the past five years, but the technique is still in very wide- spread use. Early equipment for the analysis of amino acids was very large and bulky, but newer equipment tends to be counter-top sized, and one geologist even developed a suitcase-sized instrument for use in the field. Early technology utilized stepwise gradients of buffers for elution of amino acids from the ion exchange columns, but newer instruments utilize continuous gradients, an important advance that leads to fewer baseline disturbances.
The core of the technique is the ion exchange column, composed of a hydrophilic cation exchange resin made of sulfonated polystyrene-divinylbenzene copolymer. The preparation of this resin is seemingly complex and not completely controlled, so that batch-to-batch differences can usually be detected. Separation of amino acids is seldom the result of the resin's ion exchange properties alone. Rather, partition effects, adsorption effects, and even size exclusion effects can be noted, and while these may complicate the prediction of the "expected" elution positions of individual amino acids, the reproducibility of any batch is very good and no practical complications arise. Asp (with the lowest pI) elutes early and Arg (with the highest pI) elutes late; Trp, however, doesn't elute based on its pI and is affected strongly by adsorption properties. Chromatography is done at elevated temperature, and temperature effects are very important, as are counterion and pH conditions. Na+ or Li+ are the usual counterions, and lower pH, lower ionic strength, or lower temperature all lead to increased elution volume of amino acids.
Various detection systems have been developed, with the classical one being ninhydrin. Reaction of ninhydrin with amino acids results in the development of a colored compound (Rhueman's Purple) and is responsible for the specificity of the technique. Ninhydrin does not react with all amines, and many contaminating compounds will not react with ninhydrin but would be detected by some other techniques. Recent developments include "Trione," a more stable but less sensitive derivative, and fluorescent detection reagents including fluorescamine and OPA. Utilizing OPA, it can be shown that the sensitivity of post-column techniques is approximately the same as that of the pre-column techniques, but most workers still utilize ninhydrin.
In comparing post-column and pre-column methodologies, some advantages of the post-column methods should be noted:
In addition, newer uses are being developed that may require the use of post-column derivatization. In distinguishing stereoisomeric amino acids, some workers have utilized the crown ether- derivatized cellulose chromatographic supports to separate D-Phe and L-Phe (and other amino acids). This separation may require the use of free (underivatized) amino acids; even without the newer developments, however, the use of post-column technology does not seem to be diminishing, but continues to find widespread popularity.
Pre-column derivatization is consistently used by about half of the sites that participate in the ABRF collaborative trials, and pre-column methodology is considered to be more sensitive than many of the post-column approaches. While there are several pre-column derivatization options (8), phenylisothiocyante (PITC) is by far the most common. An excellent review on PITC derivatization appeared this year (9) and may be consulted for further details. An advantage of the PITC approach compared to other pre-column methods is that PITC reacts with both primary and secondary amines, whereas some other methods will not react with proline. Other advantages include sensitivity, uv detection (as opposed to fluorescence), and stability.
After hydrolysis, samples must be dried to remove acid, and this is usually accomplished under vacuum. This leaves the amino groups in the protonated state, however, and thus less reactive to PITC. Therefore, the first step in derivatization is to "re-dry" the samples, which consists of adding a solution of ethanol, water, and triethylamine (2:2:1, v/v), followed by vacuum removal of the liquid. This results in deprotonated amines, so that when the derivatization solution is added (ethanol/water/triethylamine/PITC; 7:1:1:1 v/v), reaction proceeds rapidly (within a few minutes) to completion. Perhaps the most important step in the procedure is to remove the derivatization reagent, and this is accomplished under vacuum or by heptane extraction. We have found that a good vacuum is sufficient, but it must be < 60 mTorr for several hours (or overnight), and it is essential to have a clean coldtrap before beginning. If PITC is not removed adequately, it leads to chromatography complications and degradation of the HPLC column. Once derivatized, samples can be stored for quite some time. In the dry and frozen state, derivatized samples are stable for at least 6 months (9). After the samples are dissolved in column buffer, they are stable for at least 7 hours at room temperature (9). Chromatography is performed on C-18 reversed phase columns, and it is important to maintain a good guard column. Guard columns should be replaced every 150 runs or so, while the separation column (Waters, Pico-Tag column, 3.9 mm x 15 cm, for hydrolysates) is usually viable for 400 - 600 runs. Eluents are rather simple, usually 0.14 M sodium acetate plus 0.5 mL/L of triethylamine, pH 6.8 (buffer A) and acetonitrile: water (60:40, v/v buffer B). A linear gradient over 15 minutes plus a wash at 100% buffer B is common (9). Chromatography is performed at elevated temperature, usually 38 C (though some workers go as high as 50 C). Chromatography is most sensitive to column temperature and buffer pH; acetate concentration and TEA concentration also affect chromatography, though to a lesser extent. Typically, good resolution of 18 amino acids is obtained in 12 minutes of chromatography, not including an additional 8 minutes or so for column washing and re- equilibration.
Data from the collaborative trials of the ABRF for the last three years (1,6,7) indicate that there has been no significant difference in average error between PITC pre- column users and ninhydrin post-column users. PITC sites are often among the very best sites in terms of least error, but are also well-represented among the highest error group. It is fair to conclude that there are no major obstacles to high quality pre-column techniques, and that they can be used with accuracy as high as any other technique.
The use of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) as a pre-column derivatizing agent was first described by Strydom and Cohen (10), and has been commercialized by Waters, Inc., as AccQ-Tag. In this procedure, amino acids react with the AccQ reagent, yielding urea-type compounds. This reaction is much faster than the competing reaction of the reagent with water, and conditions for derivatization are very fast and specific. Typically, 5 uL of hydrolysate is mixed with 35 uL of borate buffer, and then 10 uL of the AQC reagent is added. Reaction requires only 1 minute, after which samples are heated for 10 min at 55 C. Chromatography occurs on a reverse phase HPLC column at 37 C using gradient elution and buffers conveniently available from Waters. Detection can be either with uv absorption or fluorescence. Modified conditions (flow rate, temperature, and solvent concentration) have been developed for microbore columns in order to decrease solvent consumption. Flow of eluents can be reduced to 0.25 mL/min, using the original gradient (10). For these columns, the optimal temperature is 35 oC but lower temperature (33 C) leads to better resolution of unmodified cystine and Tyr. Higher concentration of buffer (i.e., 110 % of that recommended by Waters) decreases resolution between Gly and His but increases resolution between Arg and Thr. Lower buffer concentration (90 %) increases resolution between Gly and His at the expense of resolution between Arg and Thr.
Advantages of this newer method include the very simple derivatization procedure, stable derivatives, excellent separation, detection by either absorbance or fluorescence, and commercial availability of reagents. Disadvantages seemingly are limited: relatively long chromatography time (57 min turn-around time) and high solvent consumption (unless microbore columns are utilized).
Since the goal of amino acid analysis is not only to identify but to quantitate each amino acid, good procedures for handling the data are required. While it is always necessary to pay careful attention to the instrumentation, reagents, and columns being employed, it is also necessary to process the data according to expected norms. This will include generating a set of standard "color values" for each amino acid and the appropriate calibration curves, followed by calculating the data in the manner most appropriate for its desired end use.
Color values and calibration curves are the most important consideration, since all values derive from them. The term color value, seemingly a remnant of earlier days of AAA, is simply the coefficient necessary to convert absorbance (or fluorescence) units to concentration or mass units, obtained by injecting known amounts of amino acid standards. Calibration curves can be developed for external or internal standards. For external standards, 3 or more concentrations of standard mixtures, and 6 or more repeats of each concentration are recommended to generate a concentration curve. Usually, if the standard values are within 3 % of the calibration values, the results will be within acceptable limits. Samples are then run in parallel with standards, and should, of course, be in the same concentration range as the standards. Many workers recommend hydrolysis of the standards, especially since fewer workers are extrapolating to obtain Ser and Thr values. Appropriate use of external standards depends on sample loading accuracy and the volumes of samples (which can be altered by desiccation during storage or other difficulties). Internal standards are also common, and should be developed by using 6 or more repeats of standards containing an added amino acid such as norleucine (i.e., one which will behave similarly to the analytes but which is found in none of the samples and is resolved from all analytes). Addition of the internal standard to the samples allows for direct reporting of amino acid concentrations in the sample. With internal standards, the key is to add the correct amount to each sample (a precision consideration).
Presentation of data can be in a variety of formats, usually depending on the users needs. Data can be shown as pmol of amino acid in the sample, as residues of amino acid per molecule (assuming the molecular weight of the molecule is known), as mole % of amino acid, or as concentration of protein in the sample. An important consideration is to report cysteine/cystine accurately: the concentration of 1/2Cys is equivalent to twice the concentration of Cys (cystine gives rise to two equivalents of cysteine or its derivative).
References
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