Amino quantitative analysis

The amino add analysis of all peptide chains on the resins indicated a ratio of Pro Val 6.6 6.0 (calcd. 6 6). The peptides were then cleaved from the resin with 30% HBr in acetic acid and chromatogra phed on sephadex LH-20 in 0.001 M HCl. 335 mg dodecapeptide was isolated. Hydrolysis followed by quantitative amino acid analysis gave a ratio of Pro Val - 6.0 5.6 (calcd. 6 6). Cycll2ation in DMF with Woodward s reagent K (see scheme below) yielded after purification 138 mg of needles of the desired cyc-lododecapeptide with one equiv of acetic add. The compound yielded a yellow adduct with potassium picrate, and here an analytically more acceptable ratio Pro Val of 1.03 1.00 (calcd. 1 1) was found. The mass spectrum contained a molecular ion peak. No other spectral measurements (lack of ORD, NMR) have been reported. For a thirty-six step synthesis in which each step may cause side-reaaions the characterization of the final product should, of course, be more elaborate. 

Note The reagent can be employed for qualitative and quantitative analysis on silica gel and RP layers. Ammonia, amine and acid-containing mobile phases should be completely removed beforehand. Amino phases cannot be employed. The NBD-chloride reagent is not as sensitive as the DOOB reagent (qv.) on RP phases. 

The inorganic sorbents act as catalysts in all this [3,4]. Hie pH also probaUy plays a role. Reactions that do not otherwise occur are observed on add silka gd [3] or basic aluminium oxide layers. Reactions of this type have also been obsoved for amino [6-8] and RP phases [9]. The products of reaction are usually fluorescent and can normally be used for quantitative analysis since the reactions are reprodudble. 

Larsen, B. R. and West, F. G., A method for quantitative amino acid analysis using precolumn o-phthaladehyde derivatization and high performance liquid chromatography, /. Chromatogr. Sci., 19, 259, 1981. 

Four different amino acids have been selected for esterification to study the effect of R-group substituent of amino acid on rate and ease of esterification. The four acids are alanine, serine, aspartic acid and lysine. Their respective esters were prepared by reported methods to authenticate and compare with those prepared by our method. Alanine was esterified with ethanol to yield the ethyl ester, keeping -NH3+ group intact. This was also confirmed by acidity of final reaction mixture (pH- 3.2). There was about 50% conversion of alanine to its ethyl ester. Further work on ester formation, including qualitative and quantitative analysis, is in process. 

Goodacre, R. Edmonds, A. N. Kell, D. B. Quantitative analysis of the pyrolysis-mass spectra of complex mixtures using artificial neural networks Application to amino acids in glycogen. J. Anal. Appl. Pyrolysis 1993, 26, 93-114. 

A detailed quantitative analysis of the preferences of amino acids in folded proteins for different regions of the Ramachandran plot reveals that the 18 nonglycine, nonproline residues exhibit different preferences (Shortle, 2002). Figure 5 shows the range of relative propensities displayed by these 18 amino acids for a somewhat arbitrary subdivision... 

S. Keck, T. Peters, Identification of protein containing paint media by quantitative amino acid analysis, Studies in Conservation, 14, 75 82 (1969). 

The column chromatography technique using Dowex 50 ion-exchange resin, introduced in 1951 (M2) and improved in 1954 (M3) by Moore and Stein, first made possible the precise quantitative analysis of amino acids liberated in the course of acid hydrolysis of urine. Similar results were also obtained by Muting in 1954 (M4), who used paper chromatography methods. In this procedure amino acids were quantitatively determined after staining on the paper and elution of the resulting spots. 

H Frank, GR Nicholson, E Bayer. Enantiomer labelling, a method for the quantitative analysis of amino acids. J Chromatog 167, 187, 1978. 

J Gerhardt, K Nokihara, R Yamamoto. Design and applications of a novel amino acid analyzer for D/L and quantitative analysis with gas chromatography, in JA Smith, JE Rivier, eds. Peptides Chemistry and Biology. Proceedings of the 12th American Peptide Symposium, Escom, Leiden, 1992, pp 457-458. 

There are several compounds that will react with amino acids to give coloured or fluorescent products and as a result can be used in qualitative or quantitative methods. Fluorimetric methods are gaining in popularity and offer some important advantages over absorption spectrophotometry for amino acid analysis. 

The identification and quantitation of the individual amino acids in a mixture is often required in metabolic studies and investigations of protein structure. The use of thin-layer chromatography or electrophoresis may be adequate to indicate the relative amounts and number of different amino acids in a sample but the use of gas-liquid chromatography or an amino acid analyser is essential for quantitative analysis. 

With methods for the quantitative analysis of amino acids to hand, the way was now open for the determination of amino acid sequences. Purified bovine insulin was relatively freely available. On the basis of ultracentrifugal analysis (Gutfreund and Ogston), a molecular weight of 12,000 was assumed—as it later emerged, a factor of 2 too high. One of the advantages from the choice of insulin as the protein to sequence was that tryptophan is absent. A 100% recovery of the amino acids could therefore be obtained easily by simple hydrolysis with HC1. In 1948 Tristram reported the complete amino acid composition of the protein. 

Moore and Stein introduced quantitative chromatographic amino acid analysis for proteins. 

There are five categories of protein assay colorimetric assays, direct absorbance methods, fluorescence methods, amino acid analysis, and custom quantitation methods. A brief summary of the principles, advantages, and limitations of these methods follows. 

Soga, T., Kakazu, Y., Robert, M., Tomita, M., andNishioka, T. (2004). Qualitative and quantitative analysis of amino acids by capillary electrophoresis-electrospray ionization-tandem mass spectrometry. Electrophoresis 25, 1964—1972. 

Problems such as diffusional limitations and the analysis of catalyst composition occur with solid-phase catalysts. Much work has been done on diffusion in bound enzymes (for reviews, see 24 and 88). In our work we used ninhydrin, which is a reagent ideal for surface analysis amino acid analysis is used wherever possible. Amine depletion as followed by ninhydrin is not exact, but some quantitative guides are obtained. Certainly synthetic catalysts must be made with bonds other than amide bonds and components other than those compounds that are detectable on the amino acid analyzer. 

Although DIOS-MS is mainly a tool for qualitative analysis, many examples have shown that quantitative analysis is possible when internal standards are used. These may either be isotope-labelled - mostly deuterated - compounds or structurally related analogues. For example, subsequent to electrospray deposition, amino acids such as phenylalanine and tyrosine have been successfully quantified by means of DIOS-MS using their deuterated analogues as internal standards. 

For the analytical characterization of sulfated tyrosine peptides, spectroscopic methods as well as amino acid analysis and, more recently, mass spectrometry are employed. In Table 2 the spectroscopic data of tyrosine 0-sulfate are compared to those of the related sulfonic acid derivatives as possible byproducts in the chemical sulfation of the tyrosine or tyrosine peptides.[361 In the course of the synthesis of tyrosine 0-sulfate peptides and, particularly in the final deprotection step, desulfation may occur which limits the characterization of the final compounds in terms of quantitative identification of the tyrosine 0-sulfate. This is achieved by amino acid analyses of basic hydrolysates of the sulfated tyrosine peptides or preferably by analyses of the enzymatic hydrolysates with aminopeptidase M or leucine-aminopeptidase. 

Among the analytical methods presently used for the characterization of natural and synthetic peptides and proteins, the primary value of amino acid analysis is the determination of absolute peptide and protein content in solids and solutions and the quantitation of their amino acid composition and stoichiometry. It involves two steps, i.e. complete hydrolysis of peptides and proteins, followed by photometric determination of the released amino adds. The steps are laborious and time-consuming, and there is a continuous need for improvement of the techniques to increase precision and sensitivity. 

Some laboratories do not have access to mass spectrometric analysis, but the number is fewer as the cost for this type of instrumentation is decreasing. It is suggested that these laboratories utilize amino acid analysis due to reduced cost and rapid turnaround. Peptide composition and stoichiometry can be determined, the technique is highly reproducible, and can be used to monitor cycle-to-cycle coupling efficiency. However, not all amino acids are recovered quantitatively. Cys and Trp are totally destroyed and must be quantitated using distinctly different hydrolysis procedures. Ser and Thr can be partially destroyed. Some laboratories perform amino acid analysis in addition to mass spectrometric analysis in order to assure peptide composition, stoichiometry, and quantity (see also Sections 7.3, 7.3.1 and 7.3.2). 

Walker V, Mills GA (1995) Quantitative methods for amino acid analysis in biological fluids. Ann Clin Biochem 32 28-57... 

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