High performance liquid chromatography peptide purification

J Rivier, R McClintock, R Galyean, H Anderson. Reversed-phase high-performance liquid chromatography preparative purification of synthetic peptides. J Chromatog 288, 303, 1984. 

The use of high performance liquid chromatography (HPLC) on-line or off-line is an essential feature for peptide mapping to integrate the removal of buffers and salts (purification) and the separation of analytes (preconcentration) with mass spectrometry. With on-line LC/MS approaches, low flow rates (<100pL/min) have been demonstrated to provide maximum sensitivity with ESI techniques for the analysis of proteins. In the work performed by Arnott and... 

Comparative assessments of purification protocols are generally difficult, and wide ranges of activities have been reported. ACV synthetases have been assayed by either employing a peptide adsorption resin (Porapak) [29,48] or high performance liquid chromatography (HPLC) detection of the derivatized tripeptide [26,27], The adsorption assay, employing radiolabeled amino acids, has been... 

Equipment and expertise. Synthesis requires dedicated laboratory space and equipment. Protein synthesis is best done with the aid of a peptide synthesizer which is capable of optimal step-wise yields. Purification using reverse-phase high-performance liquid chromatography (RP-HPLC) is an integral part of the procedure (16), so at least one preparative and one analytical HPLC systems is needed. Access to electrospray mass spectrometry is essential. 

Coppola, G., Underwood, J., Cartwright, G., and Hearn, M. T. (1989). High-performance liquid chromatography of amino acids, peptides and proteins. XCIII. Comparison of methods for the purification of mouse monoclonal immunoglobulin M autoantibodies. J. Chromatogr. 476, 269-290. 

Poon, G.K., Priestley, I.M., Hunt, S.M., Fawell, IK., andCod4 G.A. 1987. Purification procedure for peptide toxins from the cyanobacterium Microcystis aeruginosa involving high-performance liquid chromatography. Journal of Chromatography 3S7 551-555. 

The purity of crude peptide will be analayzed on a reverse-phase high-performance liquid chromatography (HPLC) column. The elution of the peptide is followed by absorbance recorded at 215 nm. The purification on a 10 to 20 mg scale is performed on a larger, preparative column monitored at the less sensitive wavelength of 238 nm. 

A stable isotope dilution assay using mass spectrometry to measure insulin, proinsulin, and C-peptide has been developed. The difference in mass among the three analytes allows specific measurement of each protein. Comparison of patient samples revealed that most, but not all, results were higher by immunoassay than mass spectrometry. Thus immunoassays may overestimate insulin, particularly at low concentrations. The high protein concentration in the serum requires extraction of proteins (e.g., by immunoaffinity) and purification by high-performance liquid chromatography (HPLC) before quantification by mass spectrometry. This method is not suitable for routine laboratory analysis. 

It is necessary to use water miscible organic solvents in order to elute proteins (Table 2-4). Most proteins are apt to be denatured in that case. RPC is useful for the purification of small samples or peptides and is usually carried out as high performance liquid chromatography (HPLC). 

The purity of protected amino acids is especially important for the synthesis of longer peptides. Standard techniques such as melting point determination, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and optical rotation are effective means of characterization. The optical purity can also be evaluated by high-performance liquid chromatography (HPLC) after derivatization with Marfey s reagent [216,217]. The advanced Marfey method refers to analysis by mass spectrometry after derivatization with Marfey s reagent [218-221]. Purification of side-chain protected amino acids by recrystallization is usually sufficient. 

Purification schemes depend on the perceived nature of the impurities. The actual experimental conditions depend on the nature of the peptide. For each technique discussed, the user is encouraged to get in touch with the manufacturers of the media, high-performance liquid chromatography (HPLC) columns, equipment, etc. to acquire technical notes, which can provide very useful information on topics such as preparing media, solvent conditions, and HPLC strategies. 

Reversed phase high performance liquid chromatography (RP-HPLC) now plays a critical role in the analysis and purification of peptides and proteins from natural and synthetic sources. The extraordinary popularity of RP-HPLC for polypeptide analysis can be attributed to a number of factors such as ... 

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