Workshop-Current Challenges in Amino Acid Analysis: Quantitation, Modified Amino Acids, and Physiological Samples.
Chaired by K. Umit Yuksel (CryoLife, Inc.).
Presentations by
Steven A. Cohen (Waters Corporation)
Lowell H. Ericsson (University of Washington at Seattle), and
Peggy Borum (Univeristy of Florida at Gainesville).
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Using Amino Acid Analysis as a Quantitative Procedure - Steven A. Cohen.
Solution concentration of proteins and peptides is often determined using general dye ligand techniques such as the Lowry or bicinchoninic acid procedures, ultraviolet spectroscopy, or total Kjeldahl nitrogen. These methods are adequate for solutions with high protein concentrations (e.g., 0.1-10.0 mg/ml), but reduced accuracy is often observed with more dilute samples. In addition, the dye techniques suffer from limited dynamic range, often requiring the analysis of several dilutions to provide useful data. In fact, all the methods, especially UV spectroscopy, can be adversely affected by common protein buffer constituents. Quantitative amino acid analysis (AAA) can be successfully used as an alternative, with marked improvement at low sample concentrations. Hydrolyzed samples are reconstituted, derivatized, and then analyzed by reversed phase HPLC. Solutions of highly purified BSA, recombinantly derived samples, and synthetic peptides have been used to demonstrate excellent linearity and reproducibility for the entire analysis including hydrolysis. The useful linear range of the procedure extends from as little as 5 mg/ml up to 5 mg/ml, thus providing three orders of magnitude linear dynamic range. Good recoveries in the presence of many buffer salts often allow derivatization without desalting. In addition to providing solution concentration, the analysis also provides a measure of sample purity by comparing the derived amino acid composition to the expected composition, thus providing confirmation of sample identity. The relative merits of the techniques will be compared, with an emphasis on common problems encountered.
Identification of Unusual Amino Acids - Lowell H. Ericsson.
It is generally accepted that all eukaryotic proteins undergo post-translational modification and frequently this involves chemical modification of an amino acid residue. In a nucleic acid sequence these modifications are usually missed, although sometimes they can be anticipated from a comparison of the translated sequence against a database of motifs. In a protein the modified residues may be rapidly identified by modern techniques in mass spectrometry. Because of possible chemical degradation of modified amino acids during the hydrolysis of a protein for amino acid analysis or degradation during Edman sequencing, many modifications may have been missed. Because amino acid analysis involves the determination of the composition of a protein, a single modified amino acid is easily missed among the large amounts of the usual amino acids. Tables of retention times of unusual amino acids on a wide variety of analytical systems have been compiled. These retention times coupled with mass spectrometry of isolated amino acids can result in identification and characterization of an unusual amino acid.
Analysis of Free Amino Acids in Physiological Samples - Peggy Borum.
Identification and quantitation of free amino acids in biological tissues and fluids are critical in metabolic basic research and in patient care of the critically ill. Several amino acids such as taurine are not found in proteins but are important constituents of the free amino acid pool. Methylation, hydroxylation, and oxidation of specific amino acids increase the number of analytes that need to be analyzed to more than 40 and thus increase the chromatographic challenges. Amino acids such as glutamine are stable in peptide linkage, but degrade rapidly as free amino acids unless special precautions are followed. Other amino acids such are cysteine are compartmentalized into three pools: i, peptide linkage; ii, no peptide linkage but disulfide linkage; and iii, free amino acids. Sample preparation procedures must begin at the moment of sample collection and be closely followed throughout sample isolation, storage, and derivatization to prevent analyte degradation or loss. Chromatographic separation using HPLC and detection of amino acid derivatives using UV/vis spectrometry or fluorometry are the techniques routinely employed today. Bench-top mass spectrometers offer new opportunities for increased sensitivity and increased specificity of amino acid analysis of physiological samples
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