Peptides chromatographic techniques

M Gain, P Lloyd-Williams, F Albericio, E Giralt. Convergent solid-phase peptide synthesis. 12. Chromatographic techniques for the purification of protected peptide segments. Int J Pept Prot Res 46, 119, 1995. 

The separation mechanism is quite different from other chromatographic techniques and a broader spectrum of possible impurities can be detected at the same system (e.g., inorganic small cations, anions by indirect detection, chiral separations by adding a chiral selector, proteins and peptides by adding a polymer to the separation buffer, etc.)... 

Unfortunately, there are numerous ways to circumvent the analysis of total amino acids, such as by the addition of ammonium salts, inexpensive amino acids, peptides and protein hydrolysates. Several approaches have been made to verify the authenticity of the total amino acid values. Rockland and Underwood (10) developed a paper chromatographic technique for quantitatively estimating the individual amino acids. 

When the ultimate objective is to produce bioactive peptides for particular purposes, such as antioxidative or antihypertensive activities, the purification of target peptides from protein hydrolysate can be carried out using UF membranes with or without chromatographic techniques. Jun et al (2004) reported successful preparation of protein hydrolysates from yellowfin sole frame by first using extracted mackerel intestine crude enzyme at pH 10.0 and 50 °C, followed by treatment with pepsin at pH 2.0 and 37 °C. The resultant hydrolysate was further fractionated through five different UF membranes with... 

A continuous improvement has allowed analysis to reach detection limits at the pico-, femto- and attomole levels [72,73], Furthermore, the direct coupling of chromatographic techniques with mass spectrometry has improved these limits to the atto- and zeptomole levels [74,75], A sensitivity record obtained by mass spectrometry has been demonstrated by using modified desorption/ionization on silicon DIOS method to measure concentration of a peptide in solution. This technique has achieved a lower detection limit of 800 yoctomoles, which corresponds to about 480 molecules [76]. 

H- and 13C-NMR data have been reported for diagnostic purposes in direct analysis of phenylthiohydantoin amino acid derivatives (PTH) produced in the Edman degradation of peptides and proteins.189-193 The insensitivity of 3H-NMR spectroscopy constitutes a major hurdle for its application in the sequence study of peptides.194,195 Alternatively, identification of the cleaved amino acids in the automated Edman degradation has been solved in some cases by using IR,196-198 mass,199 and gas chromatographic techniques.200... 

Chromatographic techniques may also reveal a range of important data. End-group analysis will give an indication of the breakdown of the peptide chain, and the measurement of extracted soluble protein will similarly indicate the extent to which the polymer has deteriorated. These techniques, alongside mass spectroscopy, can also reveal the presence of dyes and other treatments. 

For a variety of reasons, analytical determination of one or both of the optical isomers is needed. The optical methods that have been traditionally used to determine the extent of optical rotation in racemic mixtures seldom have the required sensitivity. The case in point is a typical problem of peptide synthesis where the racemization of an optical isomer may occur during the chemical reaction, and where it is highly important to know accurately the extent of such racemization. The chromatographic approach to stereoselective analyses is quite attractive resolution of the antipodes, coupled with the sensitivity of the modem chromatographic techniques, makes this approach quite unique. 

Even in favourable cases, the purification of a synthetic peptide can take considerable time and effort. The purification of protected peptide intermediates is complicated by problems of poor solubility and is often laborious. Assuming that the protected peptide can be dissolved in some suitable solvent, purification can be carried out using one or more of the chromatographic techniques that are applicable to peptide molecules. Frequently, however, quite drastic modifications of the normal operating conditions are necessary if a protected peptide is to be purified to homogeneity [10,120-122]. Two techniques in particular, deserve special mention because of their usefulness in the purification of protected peptide molecules. 

A method for cutting them specifically and reproducibly into fragments (peptides) which are small enough to be separated easily by chromatographic techniques (e.g., LC, capillary electrophoresis, 2D-TLC, electrophoresis, etc.)... 

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