Proteins reversed-phase liquid chromatography

Benedek, K., Dong, S., and Karger, B. L., Kinetics of unfolding of proteins on hydrophobic surfaces in reversed-phase liquid chromatography, /. Chromatogr., 317, 227, 1984. 

Zhou, F., Johnston, M. V. (2005). Protein profiling by capillary isoelectric focusing, reversed phase liquid chromatography, and mass spectrometry. Electrophoresis 26(7-8), 1383-1388. 

Geng, X., Regnier, F.E. (1984). Retention model for proteins in reversed-phase liquid chromatography. J. Chromatogr. 296, 15-30. 

Mao, Y., Zhang, X.M. (2003). Comprehensive two-dimensional separation system hy coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection. Electrophoresis 24, 3289-3295. 

J Vendrell, FX Aviles. Complete amino acid analysis of proteins by dabsyl derivatization and reversed-phase liquid chromatography. J Chromatogr 358 401-413, 1986. 

D. B. Wall, S. J. Berger, J. W. Finch, S. A. Cohen, K. Richardson, R. Chapman, D. Drabble, J. Brown, and D. Gostick, Continuous sample deposition from reversed-phase liquid chromatography to tracks on a matrix-assisted laser desorption/ionization precoated target for the analysis of protein digests, Electrophoresis, 23 (2002) 3193-3204. 

Macek et al. [120] developed a method to quantitate omeprazole in human plasma using liquid chromatography-tandem mass spectrometry. The method is based on the protein precipitation with acetonitrile and a reversed-phase liquid chromatography performed on an octadecylsilica column (55 x 2 mm, 3 /im). The mobile phase consisted of methanol-10 mM ammonium acetate (60 40). Omeprazole and the internal standard, flunitra-zepam, elute at 0.80 0.1 min with a total rim time 1.35 min. Quantification was through positive-ion made and selected reaction monitoring mode at m/z 346.1 —> 197.9 for omeprazole and m/z 314 —> 268 for flunitrazepam, respectively. The lower limit of quantification was 1.2 ng/ml using 0.25 ml of plasma and linearity was observed from 1.2 to 1200 ng/ml. The method was applied to the analysis of samples from a pharmacokinetic study. 

As a rule, a separation method should be used for both purification and concentration of the sample. The classic method for peptides and proteins is a reverse-phase liquid chromatography preparation of the sample, followed by a concentration step (often lyophiliza-tion) of the fraction of interest. During those steps performed on very small quantities of sample, loss on the sample can occur if care is not taken to avoid it. Lyophilization, for instance, can lead to the loss of the sample absorbed on the walls of the vial. The use of separation methods on-line with the mass spectrometer often are preferred. Micro- or nano-HPLC [32,33] and capillary electrophoresis [34], both coupled mainly to electrospray ionization/mass spectrometry (ESI-MS), are used more and more. 

J. W. Eschelbach and J. W. Jorgenson, Improved protein recovery in reversed-phase liquid chromatography by the use of ultrahigh pressures. And. Chem. 78 (2006), 1697-1706. 

The ion-exchange constant does vary from one pair of ions to another therefore selectivity can be controlled by appropriate choice of the counter ion. However, selectivity is more conveniently manipulated by controlling the pH of the mobile phase and taking advantage of differences in the values of the analytes to be separated. In contrast with reversed-phase liquid chromatography (see section 3.6.2.1) the retention of weak acids and weak bases will reach a maximum when the compounds are in their ionized forms. Zwitterionic compounds such as amino acids, peptides and proteins can be separated on anion exchangers or cation exchangers. 

The first step is the priming of the NRPS active site and a subsequent limited tryptic digest of the protein. The digested sample is loaded on a reverse-phase liquid chromatography (RPLC) C18 column, which is directly connected to the inlet of an FT mass spectrometer. During online LC separation, the eluent is analyzed by MS and MS2 on an LC timescale. In the mass spectrometer the eluent is first analyzed by broadband Fourier transform mass spectrometry (FTMS). Then, peaks in the resulting broadband FT mass spectrum are... 

Figure 4 Depiction of shot-gun proteomics using multidimensional protein identification technology a complex mixture of peptide fragments in the digest are resolved by a combination of ion-exchange and reversed-phase liquid chromatography.

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