Amino Acid, Peptide, and Protein Analytes

AMINO ACID, PEPTIDE, AND PROTEIN ANALYTES 4.6.1 Amino Acid Analytes... 

Separation of amino acids, peptides, and proteins Amino acids are interesting molecules by themselves from an analytical point of view for two reasons. They are inherently enantiomeric and are the building blocks of peptides and proteins. The separation of amino acids is usually done through a derivatization process due to the fact that the absorbance in the UV is low. The most frequently used derivatization is done by fluorescent tagging. Sensitivity can reach the subfemtomole level.136 139 Temperature control can be used to separate conformers.140 Two conformers of Tyr-Pro-Phe-Asp-Val-Val-Gly-NH2 and four conformers of Tyr-Pro-Phe-Gly-Tyr-Pro-Ser-NH2 were separated at subzero temperatures by including glycerol as an antifreeze component of the buffer. 

The ion-exchange constant does vary from one pair of ions to another therefore selectivity can be controlled by appropriate choice of the counter ion. However, selectivity is more conveniently manipulated by controlling the pH of the mobile phase and taking advantage of differences in the values of the analytes to be separated. In contrast with reversed-phase liquid chromatography (see section 3.6.2.1) the retention of weak acids and weak bases will reach a maximum when the compounds are in their ionized forms. Zwitterionic compounds such as amino acids, peptides and proteins can be separated on anion exchangers or cation exchangers. 

Capillary zone electrophoresis (CZE), micellar capillary electrokinetic chromatography (MECC), capillary gel electrophoresis (CGE), and affinity capillary electrophoresis (ACE) are CE modes using continuous electrolyte solution systems. In CZE, the velocity of migration is proportional to the electrophoretic mobilities of the analytes, which depends on their effective charge-to-hydrodynamic radius ratios. CZE appears to be the simplest and, probably, the most commonly employed mode of CE for the separation of amino acids, peptides, and proteins. Nevertheless, the molecular complexity of peptides and proteins and the multifunctional character of amino acids require particular attention in selecting the capillary tube and the composition of the electrolyte solution employed for the separations of these analytes by CZE. 

Dwyer J, ed. Analytical chromatography of amino acids, peptides, and proteins. In Franks F, ed. Protein Biotechnology. Totowa, NJ Humana Press, 1993 49-90. 

In New York, Bergmann continued his investigations of amino acids, peptides and proteins on a wider base. This was done through intensive work on analytical methods for the determination of amino acids and peptides and also through an enhanced examination of protein-cleaving enzymes with the necessary synthesis of peptides suitable for serving as model substrates. 

Chemistry and Biochemistry of the Sulphydryl Group in Amino-acids, Peptides and Proteins , by M. Friedman, Pergamon, Oxford, 1973 Sulphur Research Trends , ACS Advances in Chemistry Series No. 110,1971 The Determination of Sulphur-containing Groups , by M. R. F. Ashworth, Vol. 1, Academic Press, London, 1972 The Analytical Chemistry of Sulphur and its Compounds , ed. J. H. Karchmer, Wiley-lnterscience, New York. 1972. 

The chromatography-based separation of amino acids, peptides, or proteins has to be divided into the different applications of preparative and analytical chromatography. For analytical HPLC, the identification and quantification of single components from a low complex mixture is the most important task. Therefore, the analytes of interest have to be separated as much as possible. 

HPLC is frequently employed in the analysis of amino acids, peptides, proteins, nucleic acids, and nucleotides. HPLC is also often used to analyze for drugs in biological samples (see Workplace Scene 16.2). Due to the complex nature of the molecules to be analyzed, these techniques tend to be more complex than HPLC applications in other areas of analytical chemistry. For example, separation of nucleotides or amino acids is more difficult than testing for caffeine in beverages, even though the same instrument and same general methods would be employed. A variety of columns and mobile phases are regularly employed. 

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. 

Analytical methods used to identify monomers have improved significantly from those that quantify whole classes of compounds, such as amino acids, peptides, proteins, and primary amines (Undefriend et al., 1972) or carbohydrate-like compounds (Johnson and Sieburth, 1977) to ones that are molecule-specific (Table II). Most of these methods are based on combining chromatography techniques that can separate complex mixtures of molecules with highly sensitive detectors that can approach the nanomolar or picomolar range. Monomers are usually present at low concentrations, so... 

Once the coordinates of a molecule have been imported into RasMol, there are a number of analytical tools that can be used to visualize the many different interactions taking place within it. For instance, RasMol allows you to determine how many peptide chains there are in the protein, the positions of particular atoms within the protein, the positions of different ions and hydrophobic amino acids, alpha helices and beta sheets, the distance between two particular atoms or residues in the protein, points of contact between the protein and its ligands, the amino terminal and carboxy terminal amino acids, inter- and intramolecular disulfide bonds within the protein, and hydrogen bonds present between different atoms in the protein. 

Recently, a small molecule fluorophore phosphosensor technology referred as Pro-Q Diamond dye has been developed to detect and quantitate phosphorylated amino acids within peptides and proteins in microarrays. ° In addition to binding assays, fluorescence detection methods have also been developed for functional assays. For example, microarrays of quenched fluorescent substrates can be used to detect protease or esterase activities in the analytes. In this method, quenched fluorescent substrates are prepared by coupling the peptide substrate to coumarin, a fluorescent dye. These peptide substrates are then spotted onto the solid support... 

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