Amino acid analysis, quantitation

The concentration of EPO (6 pmol/pl) and GCSF (10 pmol/pl) were quantified by amino acid analysis. Quantitative loading of proteins onto the sequencing supports or into the SDS-gels was done using calibrated pipettes. BLG (5 pmol/ xl) was quantitatively diluted according to Applied Biosystems recommendations. HSA (1.11 pmol/gl) was used as received from Hewlett Packard. 

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. 

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. 

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. 

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. 

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. 

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. 

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... 

The Number of Tryptophan Residues in Bovine Serum Albumin A quantitative amino acid analysis reveals that bovine serum albumin (BSA) contains 0.58% tryptophan (Afr 204) by weight. 

Another useful reaction of amino side chains is that with dansyl chloride (Eq. 3-29). Many lysine derivatives can be determined quantitatively by amino acid analysis.280... 

Additional factors that may affect the reliability of the chemical scoring methods lie with the inherent difficulties of amino acid analysis. The analytical procedure for amino acid analysis can affect both the recovery and reliable quantitation of amino acids. Proteins must first be hydrolyzed to amino acids before analysis. Hydrolysis methods affect the amino acid recovery. Cystine, methionine, tryptophan, threonine, serine, and tyrosinecan bedestroyed during hydrolysis. Valine and isoleucine are released slowly and may not be completely... 

JE Hale, DE Beidler, RA Jue. Quantitation of cysteine residues alkylated with 3-bromopropylamine by amino acid analysis. Anal Biochem 216 61-66, 1994. 

RG Elkin. Quantitative amino acid analysis of feedstuff hydrolysates by reverse phase liquid chromatography and conventional ion-exchange chromatography. J Assoc Off Anal Chem 67 1024-... 

This approach has been used extensively for amino acid analysis using low-pressure ion-exchange chromatography and post-column ninhydrin reaction. Spraying, dipping and vapour-treatment techniques are well known as post-separation reactions in TLC, but these are considered only briefly since the majority of them are not quantitative. While the problems of pre-separation techniques are quite similar for TLC and HPLC, they differ considerably for post-separation reactions. 

The subunit stoichiometry for the coated vesicle enzyme has been determined by quantitative amino acid analysis (Arai et al, 1988 Xu et al, 1999). 

Today s predominance of the gas phase sequencer for sequence analysis is partly due to the advancement of PTH-amino acid analysis on HPLC, in which the analysis takes only 10 min to complete using reversed-phase column.70-72 Thus, a gas phase sequencer with an on-line PTH-analyzer can perform a single Edman degradation cycle within one hour. With the use of a microbore column and a data station which handles various data manipulations, a few pmol of PTH-amino acid can be quantitatively analyzed. Although the minimum detection level of amino acid derivatives has improved significantly in the last 10 years (103-104-fold enhancement), further improvement is expected with the use of a fluorescent compound, i.e., fluorescein isothiocyanate (FITC), and the laser detection technique. 

---