Proteins RP-HPLC

Another important development in optimizing cereal protein RP-HPLC was reported by Marchylo et al. [56]. Sterically protected wide-pore monofunctional-silane bonded Cg and CN columns (Zorbax RX-300) gave better resolution and reproducibility of gliadins and glutenin subunits than did conventional silica-based columns and were more stable. A typical separation is shown in Fig. 1. 

Many factors, such as solvent viscosity, diffusion rates, and column type, contribute to temperature effects in protein RP-HPLC. Temperature may also directly affect protein conformation or aggregation. For example, whereas fresh extracts of maize zein resolved well upon RP-HPLC [42], even at low temperature, purified zeins, especially after lyophilization and storage, separated poorly [53] (Fig. 5). Resolution was restored, however, at 70 C. These results suggest that zeins may aggregate and polymerize via hydrogen bonds between glutamine... 

Griehl, C. and Merkel, S., Synthesis and separation of pprotected tripeptide epimers by RP-HPLC, Inti. J. Peptide Protein Res., 45, 217, 1995. 

Wall, D. B. Kachman, M. T. Gong, S. Hinderer, R. Paras, S. Misek, D. E. Hanash, S. M. Lubman, D. M. Isoelectric focusing nonporous RP HPLC A two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry. Anal. Chem. 2000, 72, 1099-1111. 

FIGURE 1.2 2D liquid protein expression map of the HCT-116 human colon adenocarcinomacell line. The x-axis is in p/units from 4.0 to 7.0 in 0.2 increments. They-axis is percent B of the RP-HPLC gradient. The gray scale of the bands represents the relative intensity of each band by UV detection at 214 nm. From Yan et al. (2003) with permission of the American Chemical Society. (See color plate.)... 

These columns have been used for separation of proteins of over 200 kDa MW in our experiments as shown by analysis using a ID gel. In addition, columns with larger particle sizes have been used to separate proteins of over 400 kDa (55-56). The NPS RP-HPLC method provides a liquid phase method for separating large intact proteins for further analysis. More specifically, it provides a means of separating proteins for interfacing to mass spectrometric analysis. 

Reverse-phase HPLC (RP-HPLC) separates proteins on the basis of differences in their surface hydophobicity. The stationary phase in the HPLC column normally consists of silica or a polymeric support to which hydrophobic arms (usually alkyl chains, such as butyl, octyl or octadecyl groups) have been attached. Reverse-phase systems have proven themselves to be a particularly powerful analytical technique, capable of separating very similar molecules displaying only minor differences in hydrophobicity. In some instances a single amino acid substitution or the removal of a single amino acid from the end of a polypeptide chain can be detected by RP-HPLC. In most instances, modifications such as deamidation will also cause peak shifts. Such systems, therefore, may be used to detect impurities, be they related or unrelated to the protein product. RP-HPLC finds extensive application in, for example, the analysis of insulin preparations. Modified forms, or insulin polymers, are easily distinguishable from native insulin on reverse-phase columns. 

Although RP-HPLC has proven its analytical usefulness, its routine application to analysis of specific protein preparations should be undertaken only after extensive validation studies. HPLC in general can have a denaturing influence on many proteins (especially larger, complex proteins). Reverse-phase systems can be particularly harsh, as interaction with the highly hydrophobic stationary phase can induce irreversible protein denaturation. Denaturation would result in the generation of artifactual peaks on the chromatogram. 

S/DVB Thermal, AIBN 1-Decanol/THF 200 pm LD. capillary columns, IP-RP-HPLC of nucleic acids and RP-HPLC of proteins and peptides [49,137]... 

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