Amino acids automated analysis

The Edman reaction enabled amino acid sequence analysis to be automated. Mass spectrometry provides a sensitive and versatile tool for determining primary strucmre and for the identification of post-translational modifications. 

The 2D gel or Iso-Dalt is the most commonly used method in proteomics because of its relatively easy use, automation, high reproducibility, high resolution of proteins, and applicability to analysis by mass spectroscopy. Furthermore, protein bands separated by Iso-Dalt are readily amenable to Edman degradation or to the amino acid composition analysis. 

Waterfield M and Bridgen J (1975) Automated liquid phase sequence analysis. In Perham RN (ed.) Instrumentation in Amino Acid Sequence Analysis, pp. 41-71. London Academic Press. 

Traditionally, proteins were initially characterized by de-novo sequencing using automated Edman degradation and amino acid composition analysis. Today, however, these techniques tend to be replaced by MS, which not only provides more flexibility and sensitivity but is also amenable to the analysis of protein and peptide mixtures. Tandem mass spectrometry (MS/MS) is used for amino acid sequencing of peptides. MALDI-MS/MS is very powerful for peptide characterization and identiflcation via sequencing and sequence database searching. 

The automated amino acid analy2er depends on ion-exchange chromatography (117) and is now a routine tool for the analysis of amino acid mixtures (118). This most advanced machine can detect as Htde as 10 pmol in ninhydrin reaction analysis. One-half to two hours are required for each analysis. An analysis chart is shown in Figure 2. 

Fig. 2. Amino acid analysis by automated ion-exchange chromatography. Standard column, 4.6 mm ID x 60 mm Ninhydrin developer. Computer print out indicates retention time (RT), height and area of peaks, and the ratio of the height of an amino acid in the sample to the height of a standard amino acid.

The most widely appHed colorimetric assay for amino acids rehes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and (9-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, ia conjunction with 2-mercaptoethanol, is most often used ia post-column detection of amino acids separated by conventional automated amino acid analysis. More recently, determiaation by capillary 2one electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

Automated amino acid analysis of peptides containing asparagine (Asn) and glutamine (Gin) residues gives a peak corresponding to ammonia. Why ... 

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

Nokihara K, Gerhardt J Development of an improved automated gas-chromatographic chiral analysis system application to non-natural amino acids and natural protein hydrolysates. Chirality 2001 13 431. 

Bartok, T., Szalai, G., Lorincz, Zs., Borcsok, G., and Sagi, F., High-speed RP-HPLC/FL analysis of amino acids after automated two-step derivatization with o-phthaldialdehyde/3-mercaptoproprionic acid and 9-fluorenylmethyl chloroformate, /. Liq. Chromatogr., 17, 4391, 1994. 

Villanueva, V. R. and Adlakha, R., Automated analysis of common basic amino acids, mono-, di-, and polyamine phenolic amines, and indoleamines in crude biological samples, Anal. Biochem., 91, 264, 1978. 

Although the free amino acids are present only at very low concentrations in oceanic waters, their importance in most biological systems has led to an inordinate amount of effort toward their determination in seawater. A sensitive, simple, and easily automated method of analysis, the colorimetric nin-hydrin reaction, has been known in biochemical research for many years. In order for the method to be useful in seawater, the amino acids had to be concentrated. This concentration was usually achieved by some form of ion exchange [251]. An automated method not using a concentration step was developed by Coughenower and Curl [252]. While the method was used successfully in Lake Washington, its limit of detection (0.5 imol/l) is just too great for most oceanic samples. 

Webb KL, Wood L (1967) Improved techniques for analysis of free amino acids in seawater. In Scova NB et al. (eds) Automation in analytical chemistry. Technicon Symposium 1966, Vol. 1. Mediad Inc., New York pp 440-444... 

Although this technique is relatively straightforward and automated amino acid analysers are commercially available, it is subject to a number of disadvantages that limits its usefulness in bi-opharmaceutical analysis. These include ... 

Certainly, a vast amount of experience has been gained by the widespread use of conventional amino acid analysers. They offer high reliability, accuracy, reproducibility and can separate complex samples. Because conventional analysers can be fully automated, they are widely used in routine analysis. However, the method is limited by the sensitivity which can be achieved using ninhydrin as the derivatizing agent. Sensitivity can be increased by using ortho-phthaldialdehyde (OPA) instead, but where extremely high sensitivity is required, HPLC is the method of choice. 

Reaction with phenylisothiocyanate (PITC) in alkaline conditions produces stable phenylthiocarbamyl (PTC) adducts which can be detected either in the ultraviolet below 250 nm or electrochemically. However, this method involves a complex derivatization procedure and offers poorer sensitivity than the alternatives available for individual amino acids. It is useful, however, in conjunction with the automated analysis of peptides when single derivatized residues can be cleaved and analysed after conversion in acidic conditions to phenylthiohydantoins. 

A study involving twenty-six laboratories was carried out to assess the quality of amino acid analysis, using samples of urine and lyophilized plasma. Coefficients of variation ranged from 13% for glycine to 65% for methionine. Automated IEC followed by ninhy-drin detection (37) seemed to perform better than other methods however, there was no clearly superior method and no analyzer clearly outperformed the others. This seems to point to the importance of personal proficiency and expertise in the performance of such analyses137. 

NDA derivatization has also been automated for analysis of amino acids in brain tissue and microdialysates (Shah et al, 1999). NDA reacts with primary amines in the presence of cyanide to form a highly stable N-substituted l-cyanobenz[/] isoindole (GBI) derivative. Addition of a nucleophile, such as cyanide, hydrogen sulphite, isothiocyanate, or 2-mercaptoethanol, is essential for the formation of the derivative. 

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