Protein reverse-phase chromatography

Acetonitril is often recommended because of its separating power (cf. Fig. 5). Adding a small amount of trifluoroacetic acid (TFA) to both gradient components A and B, is state of the art in protein reversed-phase chromatography. It decreases retention time, improves resolution, and increases the recovery of the sample proteins. 

Here again, not all chromatographic setups are usable for any proteomics question. The use of protein reverse phase chromatography, which has been advocated for plasma proteomics (Moritz et al. 2005), precludes in turn the use of any detergent of any type. This prevents the use of this chromatographic setup in most subcellular proteomics experiments, where detergents must be used to solubilize the membrane limiting the subcellular compartments. 

Reversed-phase chromatography is widely used as an analytical tool for protein chromatography, but it is not as commonly found on a process scale for protein purification because the solvents which make up the mobile phase, ie, acetonitrile, isopropanol, methanol, and ethanol, reversibly or irreversibly denature proteins. Hydrophobic interaction chromatography appears to be the least common process chromatography tool, possibly owing to the relatively high costs of the salts used to make up the mobile phases. 

Whereas recombinant proteins produced as inclusion bodies in bacterial fermentations may be amenable to reversed-phase chromatography (42), the use of reversed-phase process chromatography does not appear to be widespread for higher molecular weight proteins. 

Reversed-phase chromatography rehes on significantly stronger-hydrophobic interactions than in HIC, which can result in unfolding and exposure of the interior hydrophobic residues, i.e., leads to protein denaturation and irreversible inactivation as such, RPC depends... 

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

Many process mixtures, notably fermentations, require sample preconcentration, microdialysis, microfiltration, or ultrafiltration prior to analysis. A capillary mixer has been used as a sample preparation and enrichment technique in microchromatography of polycyclic aromatic hydrocarbons in water.8 Microdialysis to remove protein has been coupled to reversed phase chromatography to follow the pharmacokinetics of the metabolism of acetaminophen into acetaminophen-4-O-sulfate and acetaminophen-4-O-glucu-ronide.9 On-line ultrafiltration was used in a process monitor for Aspergillus niger fermentation.10... 

Lau, S. Y. M., Taneja, A. K., and Hodges, R. S., Effects of high-performance liquid chromatographic solvents and hydrophobic matrices on the secondary and quaternary structure of a model protein. Reversed-phase and size exclusion high-performance liquid chromatography, /. Chromatogr., 317, 129, 1984. 

Figure 2.6. LC-tandem mass spectrometry to examine complex mixtures. The mixture of many different proteins is digested to yield peptides and the peptides are resolved into fractions hy cation exchange chromatography followed by reverse phase chromatography. The fractionation steps resolve the peptides into fractions that he processed hy tandem mass spectrometry to yield sequence information suitable for database searching.

Ducret, A., Van Oostveen, I., Eng, J.K., Yates, J.R., 3rd, Aebersold, R. (1998). High throughput protein characterization by automated reverse-phase chromatography/electrospray tandem mass spectrometry. Protein Sci. 7, 706-719. 

These systems rely on various combinations of size-exclusion chromatography, reversed-phase chromatography, and zone electrophoresis to characterize amines, peptides, and proteins (Yamamoto etal., 1989 Bushey and Jorgenson 1990 Larmann et al., 1993, Moore and Jorgenson, 1995 Optick and Jorgenson, 1997). Haleem Issaq reviews these separations in Chapter 16 of this book. 

Purify the biotinylated protein or molecule using dialysis or gel filtration. For small molecule biotinylation where these separation methods may not be appropriate, other procedures may have to be developed, such as reverse-phase chromatography or organic precipitation techniques. 

Reverse-phase chromatography may also be used to separate proteins on the basis of differential hydrophobicity. This technique involves applying the protein sample to a highly hydrophobic column to which most proteins will bind. Elution is promoted by decreasing the polarity of the mobile phase. This is normally achieved by the introduction of an organic solvent. Elution conditions are harsh and generally result in denaturation of many proteins. 

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