HPLC analysis of peptides

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,... 

The elevated mechanical and thermal stabilities of pellicular stationary phases having a solid, fluid-impervious core have favored the development of nonporous particles tailored for the rapid HPLC analysis of peptides, proteins, and other biopolymers. Most of these applications are in RPC. 

In 1990, Bushey and Jorgenson developed the first automated system that eoupled HPLC with CZE (19). This orthogonal separation teehnique used differenees in hydrophobieity in the first dimension and moleeular eharge in the seeond dimension for the analysis of peptide mixtures. The LC separation employed a gradient at 20 p.L/min volumetrie flow rate, with a eolumn of 1.0 mm ID. The effluent from the ehromatographie eolumn filled a 10 p.L loop on a eomputer-eontrolled, six-port miero valve. At fixed intervals, the loop material was flushed over the anode end of the CZE eapillary, allowing eleetrokinetie injeetions to be made into the seeond dimension from the first. 

Peterson, J. A., Lorenz, L. J., Risley, D. S., and Sandmann, B. J., Amino acid analysis of peptides using HPLC with evaporative light scattering detection, /. Liq. Chromatogr. Related Technol., 22, 1009, 1999. 

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...

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]...

The application of high-performance capillary zone electrophoresis (HP-CZE) in its various selectivity modes has become a very valuable adjunct to HPLC for the analysis of peptides. For synthetic peptides, in particular, both HPLC and HP-CZE now form essential components of the analytical characterization of these molecules. Increasingly, zonal, micellar, or (biospecific) affinity-based HP-CZE procedures with open tubular capillary systems are adapted to allow resolution with extremely high separation efficiencies (e.g., >105 plates per meter) of synthetic or naturally occurring peptides as part of the determination of their structural, biophysical, or functional properties. Illustrative of these capabilities are the results shown in Figure 19 for the separation of several peptides with different charge and Stokes radius characteristics by HP-CZE. 

Table 5 A Comparison of CZE, p-HPLC, and CEC Procedures for the Analysis of Peptides...

Of the 20 respondents, 19 use mass spectrometry for routine analysis of peptides, 16 use HPLC, and seven use amino add analysis. 

The greatest advance in the analysis of peptides has been the coupling with spectrometric techniques, both for identification and for characterization. Their high accuracy, sensitivity, and, in some instances, tolerance to solvents make mass spectrometers ideal detectors for analyses of HPLC-separated peptides. 

One of the problems attaching to the HPLC separation of peptides is the analysis of stereoisomers (enantiomers and diastereoisomers), that is, of peptides that differ only in the configuration of their amino acid residues. 

Many groups have used electrophoresis to enhance a primary chromatographic separation. These techniques can be considered to be two-dimensional, but they are not comprehensive, usually due to the loss of resolution in the interface between the two methods. For instance, capillary electrophoresis was used in 1989 by Grossman and co-workers to analyze fractions from an HPLC separation of peptide fragments. In this study, CE was employed for the separation of protein fragments that were not resolved by HPLC. These two techniques proved to be truly orthogonal, since there was no correlation between the retention time in HPLC and the elution order in CE. The analysis time for CE was found to be four times faster than for HPLC (12), which demonstrated that CE is a good candidate for the second dimension in a two-dimensional separation system, as will be discussed in more detail later. 

Amino acids are derivatized two ways to increase sensitivity. Free amino acids in solution are reacted with o-phthaldehyde (OPA) to form a fluorescent derivative that excites at UV,230nm, and emits at FL, 418 nm. These OPA derivatives are separated on Ci8 in a complex mixture of An/MeOU/ DMSO/water at pH 2.65. PTH amino acids are formed from the N-terminai end of peptides during Edman degradation for structure analysis of peptides and proteins. HPLC is used to identify which amino acids are released. PTH amino acids are separated at UV, 254 nm, on a Ci8 column with a gradient from 10% THF/water containing 5 mM acetic acid to 10% THF/AN.The separation with reequilibration takes 60min. Work with short 3-pm columns has reduced this separation to a 10-min gradient. 

The introduction of "fast HPLC" has proven to be particularly valuable in protein analysis. As stated earlier, assay time in RP-HPLC analysis of proteins is typically long compared to that for smaller organic molecules. We have evaluated the use of 0.6-cm ID x 4-cm columns packed with 3-um particles in the analysis of insulin by RP-HPLC for potency determination, related substances, and in peptide mapping (7). The use of the "fast column" allows considerable savings (40-60%) in analysis time, compared to the regular (0.46-cm ID x 25-cm) columns, without loss in resolving power. 

RAPID ANALYSIS OF PEPTIDES AND PROTEINS. Columns packed with micropellicular stationary phases can be utilized in many areas of protein chemistry and biotechnology where high analytical speed and resolution are required. Peptide mapping by HPLC is routinely employed for the determination of structure of proteins (2, 3,32). 

Minano FJ, Peinado JM, Myers RD (1989) Neurotensin perfused in hypothalamus of sated or fasted rat HPLC analysis of release of DA, NE and 5-HT and their metabolites. Peptides 9 1381-1387. 

N-alkyl silica-based stationary phases typically employed in HPLC separation of peptides and proteins have been investigated to a large extent in CEC mode. In most cases, gradient elution was required to optimize the resolution and analysis time. Tryptic digests maps of cytochrome c by pressurized CEC were better resolved than by pHPLC [93], The experiments were conducted at low pH in the presence of trifluoroacetic acid (TFA) to prevent tailing of basic peptides, on a column packed with 1,5-pm C18 silica particles. It has been... 

Figure 9.27 Isocratic and gradient HPLC analysis of median eminence (ME) supernatant fraction after incubation with LHRH. Total peptidase activity was assessed in ME and was analyzed by HPLC. Upper HPLC tracing represents a preboiled control, the middle tracing a zero-time incubation, and the lower a 2-hour preincubation. The peptide fragments are indicated in the upper panel by letter (A-D) and the corresponding peaks they represent are shown by the same letter. (From Advis et al., 1982.)...

Sequencing of Romalea PDF was straight forward, and it also proved to be an octadecapeptide (40). The Romalea peptide differs from Acheta PDF at a single position, the former having Leu-14 in place of Val-14. In these peptides the presence of Asp-Ala bond, positions 17-18, permitted the cleavage of the C-terminal residue by heating in dilute acid. HPLC analysis of the derivatized cleaved product enabled the identification of the C-terminus as Ala-amide. 

P. Palma and A. CappieUo, Micro-HPLC split flow gradient elution in the analysis of peptides, Annali di Chinica, 82 (1992), 371. 

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