Analysis peptide mixture, HPLC

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. 

HPLC fractionation is the first step (dimension) in the two-dimensional analysis of complex peptide mixtures. This step is generally carried out by gradient elution... 

MALDI and ESI represent the predominant ionization techniques in mass spectrometry-based proteomics, as recognized by the Nobel Prize in chemistry in 2002. MALDI is mainly used to volatize and ionize simple polypeptide samples for mass spectrometric (MS) analysis at high speed. The analysis of more complex peptide mixtures is usually conducted via ESI mass spectrometry (ESI MS) coupled online with a high-pressure liquid chromatography (HPLC) system to concentrate and separate peptides prior to MS analysis. 

ESI mass spectrometry was performed on approximately 32 pmol of peptide mixture in decylglucopyranoside buffer without success (data not shown). PVDF-bound transferrin was digested in lower concentrations of detergent, and substitution of ammonium bicarbonate for Tris, although these conditions are optimum for ESI mass spectrometric analysis the digestion was not successful as determined by HPLC analysis (7). 

Although the best HPLC method for peptide analysis is RPC, HIC offers a different selectivity for those peptides possessing three-dimensional conformations under high-salt conditions. When the separations of peptide mixtures by HIC and RPC have been compared, peaks were generally narrower on RPC due to the organic mobile phase. In a study of calcitonin variants, it was seen that peptides with certain amino acid substitutions could not be resolved by RPC, but were separated by HIC [II]. The main utility of HIC for peptide separations seems to lie in applications for extremely hydrophilic or hydrophobic peptides, or those with three-dimensional structures stable in high salt. 

The scheme summarises all modern analytical and preparative chromatography protocols, such as high-performance liquid chromatography (HPLC) and gas-liquid chromatography (GLC), with all their conceivable variations. Reverse-phase HPLC or GLC , in which a non-polar liquid is adsorbed onto the solid - the stationary phase - is more appropriate for the analysis of mixtures of derivatives of amino acids and peptides. Cellulose in the above scheme would be replaced by a less-polar medium, such as acetylated cellulose, silanised silica gel, etc. in standard reversed-phase HPLC. 

The techniques have their uses for rapid and simple monitoring of mixtures to determine the approximate relative amounts of components. Preparative TLC is often useful to purify the product of a small-scale synthesis (e.g. 0.25 mm silica gel layers and elution of peptides with a 6 3 1 mixture of EtOAc MeOH water as the mobile phase, to isolate 4-10 mg of a peptide product). Attempts to make the method more sophisticated, to give reliable quantitative information, have been largely unsuccessful. Perhaps the simplification and wide availability of HPLC techniques have suppressed interest in furthering the role of TLC for analysis of mixtures of amino acids, but improved stationary phases have contributed to better reproducibility (HPTLC), and routine TLC monitoring to validate the purity of intermediates in peptide syntheses is widely used (Barlos et al., 1993). 

Caprioli, R.M. DaGue, B. Fan, T. Moore, W.T. Microbore HPLC/mass spectrometry for the analysis of peptide mixtures using a continuous flow interface. Biochem.Biophys.Res.Commun., 1987, 146, 291-299 [LC-MS microbore gradient UV detection cow sheep pig horse]... 

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