Peptides, HPLC analysis

Figure 12.9 Overview of the production of Refludan (recombinant hirudin). The exact details of many steps remain confidential for obvious commercial reasons. A number of QC checks are carried out on the final product to confirm the product s structure. These include amino acid composition, HPLC analysis and peptide mapping...

Most HPLC instruments monitor sample elution via ultraviolet (UV) light absorption, so the technique is most useful for molecules that absorb UV. Pure amino acids generally do not absorb UV therefore, they normally must be chemically derivatized (structurally altered) before HPLC analysis is possible. The need to derivatize increases the complexity of the methods. Examples of derivatizing agents include o-phthaldehyde, dansyl chloride, and phenylisothiocyanate. Peptides, proteins, amino acids cleaved from polypeptide chains, nucleotides, and nucleic acid fragments all absorb UV, so derivatization is not required for these molecules. 

The DCL was created under standard disulfide exchange conditions at pH 7.5. After equilibration for 48 hours, each of the expected 15 disulfide products could be observed in the library using LC-MS analysis (Fig. 2.11). The library was then equilibrated in the presence of CaM, followed by a centrifugation filtration step to separate protein/bound components from free components in solution. Analysis of the filtrate was complicated by the filtration membrane affecting the composition of the library. The bound components, however, provided meaningful results. Denaturation and filtration afforded a mixture of all peptides that had bound to CaM in the course of the DCL. HPLC analysis indicated significant amplification of dimer cc (80%) and a small amplification of dimer ec (10%). Resynthesis of these two components and binding assay established values of 10 and... 

Martin, S. E., Shabanowitz, J., Hunt, D. R, and Marto, J. A., Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI Eourier transform ion cyclotron resonance mass spectrometry, Analytical Chemistry 72(18), 4266 274, 2000. 

In another study, Kalghatgi et al. [36] looked at the purification of mellittin, a 26-amino-acid peptide derived from bee venom, using micropellicular C18 stationary phases. Rapid HPLC analysis (1 min acetonitrile gradient at 80°C) of a commercially derived melittin sample revealed a complex mixture (Figure 11.14). 

HPLC analysis of food proteins and peptides can be performed for different purposes to characterize food, to detect frauds, to assess the severity of thermal treatments, etc. To detect and/or quantify protein and peptide components in foods, a number of different analytical techniques (chromatography, electrophoresis, mass spectrometry, immunology) have been used, either alone or in combination. The main advantages of HPLC analysis lie in its high resolution power and versatility. In a single chromatographic run, it is possible to obtain both the composition and the amount of the protein fraction and analysis can be automated. 

Fig. 6 Dynamic combinatorial peptide library that expioits enzyme reactions to control self-assembly processes under thermodynamic controi. (a) Emergence of the potentiai peptide derivatives of varying length in a library of interconverting molecules formed from the staring materials of Fmoc L/L2 system. Fmoc-Ls is preferentially formed. Corresponding AFM images of the fibrillar structures formed at 5 min after the addition of enzyme, and the sheet-like structures observed after 2000 h show that redistribution of the derivatives is accompanied by the remodelling from fibres (Fmoc L3) to sheet-like structures (Fmoc L5). (b) HPLC analysis of the composition of the system reveals the formation and the stabilisation of Fmoc-Ls over time. Modified from [21]...

Thiazole-containing linear peptides have also been isolated from tunicates. Virenamides A-E (412-416) were obtained from the didemnid tunicate Diplosoma virens, which contains symbiotic prokaryotic algae in its cloacal cavity. The structures of virenamides A-C were determined by HPLC analysis using Marfey s procedure [323] while the absolute stereochemistry of virenamide E (416) was proven by its synthesis from virenamide A (412) [324], Compounds 412-416 showed modest cytotoxicity toward a panel of cultured cells and 412 also exhibited topoisomerase II inhibitory activity. 

Gonzalez de Llano et al. (47) separated amino acids from low-molecular-weight peptides by means of size-exclusion chromatography on Sephadex G-10, with water as the solvent, as a preparatory step before RP-HPLC analysis of peptides from blue cheeses soluble in 5% PTA (Fig. 1). This technique has also been used (51a) to eliminate the amino acids from the ethanol-... 

V. SEPARATION MECHANISMS USED IN HPLC ANALYSIS OF PEPTIDES A. Reversed-Phase (RP) Chromatography... 

The detection of cow s milk in ewe s or goat s milk and cheese is yet another application of the HPLC analysis of peptides. Tobler et al. (125) used HPLC to examine the differences between the caseins in the milks of various species. Goat s- and cow s-milk cheese caseins were hydrolyzed with trypsin, and the peptides thus obtained were separated by reversed-phase HPLC. The chromatograms for the caseins of each species were reproducible and distinct. Subsequently,... 

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