HPLC peptide research

The rapid advancement in peptide research over the past 25 years must be attributed, in part, to the effectiveness of high-performance liquid chromatography (HPLC), particularly reversed-phase chromatography, in the separation and analysis of peptides. The resolution and selectivity of this technique allows peptides to be effectively isolated and purified from closely related substances. It also separates most or all of the components of complex biological mixtures such as tryptic digests of proteins. 

K McMellop, W Davidson, G Hansen, D Freeman, N Pallai. The characterization of crude products from solid-phase peptide synthesis by v-HPLC/fast atom bombardment mass spectrometry. Peptide research 4 40-46, 1991. 

Le Bihan, T., Robinson, M. D., Stewart, 1. L, and Figeys, D., Definition and characterization of a trypsinosome from specific peptide characteristics by nano-HPLC-MS/MS and in silico analysis of complex protein mixtures. Journal of Proteome Research 3(6), 1138-1148, 2004. 

Danilo Corradini is research director at the Institute of Chemical Methodologies of the Italian National Research Council (CNR) and a member of the General Scientific Advisory Board of CNR. His involvement in separation science started in 1976 with his research work on chromatography and electrophoresis for his PhD studies in chemistry, which was carried out at Sapienza University of Rome, Italy, under the direction of Michael Lederer, founder and first editor of the Journal of Chromatography. In 1983-1984, he worked with Csaba Horvath, the pioneer of HPLC, at the Department of Chemical Engineering at Yale University, New Haven, Connecticut, where he initiated his first investigations on the HPLC of proteins and peptides, which he continued at the Institute of Chromatography of CNR after he returned to Italy. 

The purpose for the 1998 study was to assess the ability of and determine the means whereby member facilities identify and solve problems in peptide synthesis 10 The potential for oxidation of amino acids such as methionine is always a concern for peptide chemists and biomedical researchers. A peptide mixture containing 70% correct peptide and 30% oxidized peptide was prepared and sent to member facilities to determine if the oxidized methionine would be detected (see Table 1). In addition to the oxidized peptide, a reverse synthesized peptide was sent to the participants. In previous studies, peptides had been submitted which had been synthesized in the reverse order and if only HPLC and mass spectrometric analyses was performed, the reverse synthesis would not be identified. Therefore, two peptides were designed with the second in the reverse order with two substitutions to equal the mass of the first peptide. These two peptides were readily separated by HPLC. The second peptide was sent to the laboratories, but the laboratories were given the first sequence and asked if the correct peptide had been made. Out of 20 participating laboratories ... 

Unfortunately, most flavor researchers have been slow to utilize the speed and sensitivity that HPLC offers. Although bitter amino acids and peptides have been reported in cheese and casein (36, 37) and in yoguart (38) all the analyses were done by traditional methods. 

LC/MS analysis of proteins and peptides is an important part of drug discovery process, as illustrated in this chapter. The combination of various HPLC techniques and advanced MS methods provides unique analytical capabilities of structural identifications for therapeutic proteins and target proteins. The continuous evolution of proteomics research provides both an opportunity and a challenge for further developments in separation techniques and MS characterization methods. It is expected that these analytical techniques will continue to play important roles in drug discovery in the future. 

Recombinant human TGF-a was provided by Dr. R. N. Harkins (Berlex Biosciences, Inc. Richmond, CA). The methods of expression, harvesting, refolding and purification have been previously described (5 and references therein). EGFR-ED was provided by Dr. Maureen O Connor-McCourt (Biotechnology Research Institute, Montreal, PQ). Expression and purification using this system has been described previously (5 and references therein). Desmopressin was synthesized by solid-phase peptide synthesis methods and was purified by reversed-phase HPLC (6). Pure bovine neurophysin-II (NP-II) was provided by Dr. Esther Breslow (Cornell University, Ithaca, NY). 

Sample and Solvents. Samples of peptides and pharmaceuticals were obtained from Sigma Chemical Co. (St. Louis. MO) and the pesticides were obtained from the EPA repository (Research Triangle Park, NC) and were used as received. Standard solutions (1 mg/mL) were prepared with HPLC grade methanol (Burdick and Jackson, Muskegon, MI). Dilution of the standard solutions down to 100 ng/mL were performed to determine detection limits. All solutions were stored in a freezer when not in use. 

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