HPLC. amino acid

MC Aristoy, F Toldra. Deproteinization techniques for HPLC amino acid analysis in fresh pork muscle and dry-cured ham. I Agric Food Chem 39 1792-1795, 1991. 

U Butikofer, D Fuchs, JO Bosset, W Gmur. Automated HPLC-amino acid determination of protein hydrolysates by precolumn derivatization with OPA and FMOC and comparison with classical ion exchange chromatography. Chromatographia 31 441-447, 1991. 

Peptide mapping, HPLC, IEF, mass spectrophotometry, amino acids sequencing Peptide mapping, HPLC, mass spectrophotometry IEF, SDS-PAGE, HPLC, amino acids sequencing SDS-PAGE, HPSEC... 

Chloroacetylated oligotuftsin H-[Thr-Lys-Pro-Lys(ClAc)-Gly]4-NH2 was dissolved in 0.1 M Tris-HCl buffer (pH 8.2) in a peptide concentration of 1 mg/ mL. The 20-mL tube used for the reaction was filled up fully and closed tightly. The epitope peptide synthesized by the procedure presented in Subheading 3.1.1. (H- LKN1cADPNRFRGKDL22C-NH2) was added to the mixture in 1-2 mg aliquots every 15 min (see Note 5). In this way low concentrations of the epitope peptide prevented disulfide bond formation. The coupling was followed by analytical HPLC that showed the incorporation of the epitope peptide. The reaction was completed in 2 d using two equivalents of epitope peptide to each chloroacetyl group (see Note 6). The branched chimeric peptide was purified on RP-HPLC (60% yield) (see Note 7) and after lyophilization the purified product was characterized by analytical HPLC, amino acid analysis and mass spectrometry. 

Proteias, amino acids bonded through peptide linkages to form macromolecular biopolymers, used as chiral stationary phases for hplc iaclude bovine and human semm albumin, a -acid glycoproteia, ovomucoid, avidin, and ceUobiohydrolase. The bovine semm albumin column is marketed under the name Resolvosil and can be obtained from Phenomenex. The human semm albumin column can be obtained from Alltech Associates, Advanced Separation Technologies, Inc., and J. T. Baker. The a -acid glycoproteia and ceUobiohydrolase can be obtained from Advanced Separation Technologies, Inc. or J. T. Baker, Inc. 

These methodologies have been reviewed (22). In both methods, synthesis involves assembly of protected peptide chains, deprotection, purification, and characterization. However, the soHd-phase method, pioneered by Merrifield, dominates the field of peptide chemistry (23). In SPPS, the C-terminal amino acid of the desired peptide is attached to a polymeric soHd support. The addition of amino acids (qv) requires a number of relatively simple steps that are easily automated. Therefore, SPPS contains a number of advantages compared to the solution approach, including fewer solubiUty problems, use of less specialized chemistry, potential for automation, and requirement of relatively less skilled operators (22). Additionally, intermediates are not isolated and purified, and therefore the steps can be carried out more rapidly. Moreover, the SPPS method has been shown to proceed without racemization, whereas in fragment synthesis there is always a potential for racemization. Solution synthesis provides peptides of relatively higher purity however, the addition of hplc methodologies allows for pure peptide products from SPPS as well. 

Gas Chromatography (gc). A principal advantage of gas chromatography has been the faciUty with which it can be combined with mass spectrometry for amino acid identification and confirmation of purity. The gc-mass spectrometry combination offers the advantage of obtaining stmctural information rather than the identification by retention time in hplc. 

Polypeptides. These are a string of a-amino acids usually with the natural 5(L) [L-cysteine is an exception and has the R absolute configuration] or sometimes "unnatural" 7f(D) configuration at the a-carbon atom. They generally have less than -100 amino acid residues. They can be naturally occurring or, because of their small size, can be synthesised chemically from the desired amino acids. Their properties can be very similar to those of small proteins. Many are commercially available, can be custom made commercially or locally with a peptide synthesiser. They are purified by HPLC and can be used without further purification. Their purity can be checked as described under proteins. 

Electrostatic effects have long been recognized in commercial HPLC columns for SEC of proteins (15,21,22). The usual remedy is to add 100 mM salt to the mobile phase. This works here too the Lys and Asp peaks collapse into the Gly peak with 100 mM salt (Eig. 8.8). High concentrations of sodium sulfate were added to determine the role played in SEC by hydrophobic interactions (sodium sulfate, a structure-forming salt, strengthens such interactions). Sodium sulfate increased the retention only of the most hydrophobic amino acids to any extent, and then only when the concentration approached 1 M. Clearly, hydrophobic interaction cannot account for the elution order of amino acids on PolyHEA. 

FIGURE 4.22 HPLC chromatogram of amino acids employing precolumn derivatiza-tion with OPA. Chromatography was carried out on an Ultrasphere ODS column using a complex tetrahydrofuran methanol 0.05 M sodium acetate (pH 5.9) 1 19 80 to methanol 0.05 M sodium acetate (pH 5.9) 4 1 gradient at a flow rate of 1.7 mL/min. 

However, the use of a HPLC separation step enabled a remarkable acceleration of the deconvolution process. Instead of preparing all of the sublibraries, the c(Arg-Lys-O-Pro-O-P-Ala) library was fractionated on a semipreparative HPLC column and three fractions as shown in Fig. 3-2 were collected and subjected to amino acid analysis. According to the analysis, the least hydrophobic fraction, which eluted first, did not contain peptides that included valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine residues and also did not exhibit any separation ability for the tested racemic amino acid derivatives (Table 3-1). 

Table 3-1. Values of enantiomeric resolution of DNP-amino acids in a running electrolyte containing the three fractions 1, 2, and 3 of the cyclo(Arg-Lys-X-Pro-X-(3 Ala) sublibrary separated by preparative HPLC.

As expected from the design of the experiment, the HPLC column packed with CSP 14 containing all 36 members of the library with tt-basic substituents separated 7t-acid substituted amino acid amides. Although encouraging since it suggested the presence of at least one useful selector, this result did not reveal which of the numerous selectors on CSP 14 was the most powerful one. Therefore, a deconvolution process involving the preparation of series of beads with smaller numbers of attached selectors was used. The approach is schematically outlined in Fig. 3-17. 

Amino acid sequencing may be carried out in a number of ways. The most widely used is the Edman degradation procedure in which phenylisothiocyanate is used to react with the amino acid residue at the amine end of the protein chain. This derivatized residue is removed from the remainder of the protein and converted to a phenylhydantoin derivative which is identified by using, for example, HPLC. 

The stability of the CBI derivative is sufficient for its isolation and complete characterization (11), an accomplishment that is not realized with most OPA adducts. Thus, the CBI derivatives of a number of representative amino acids and amines have been isolated and their fluorescent properties determined as a function of the media and other relevant parameters encountered in reverse-phase HPLC (RP-HPLC). 

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