Protein HPLC resolution enhancement

Several studies have since shown, however, that selective precipitation or extraction of glutenin subunits, based on their different solubilities in aqueous alcohol solutions, is useful to fractionate reduced glutenin into alcohol-soluble (low-MW) and -insoluble (high-MW) subunits [30]. This can significantly enhance RP-HPLC resolution because analytical conditions for each protein type can be optimized [31-33]. 

Many groups have used electrophoresis to enhance a primary chromatographic separation. These techniques can be considered to be two-dimensional, but they are not comprehensive, usually due to the loss of resolution in the interface between the two methods. For instance, capillary electrophoresis was used in 1989 by Grossman and co-workers to analyze fractions from an HPLC separation of peptide fragments. In this study, CE was employed for the separation of protein fragments that were not resolved by HPLC. These two techniques proved to be truly orthogonal, since there was no correlation between the retention time in HPLC and the elution order in CE. The analysis time for CE was found to be four times faster than for HPLC (12), which demonstrated that CE is a good candidate for the second dimension in a two-dimensional separation system, as will be discussed in more detail later. 

The prefractionation technique provides improvements in separation resolution, increased sensitivity, and enhanced ability of loading a much higher amount of sample in any narrow pH interval in gel electrophoresis. Once the complexity of proteins is reduced by prefractionation, individual fractions can be subjected to either 2D gel electrophoresis or multidimensional HPLC for further separation, followed by MS analysis. 

Chromatography in all its formats is capable of resolving a simple mixture using one procedure. However the resolution of a complex mixmre requires the use of two orthogonal procedures. Two-dimensional TLC, GC, and HPLC have been used for the separation of plant extracts, environmental samples, and cell and semm protein digests to mention a few. Many approaches have been employed in which the first dimension is fractionation (to simplify the complexity of the mixture) while the second dimension is used to enhance the separation of the fractions. 

Singh et al. [40] also used an SDS-containing solvent to fractionate wheat proteins by SE-HPLC on silica-based columns, but column performance deteriorated quite rapidly [60]. The alternative solvent of 50% aqueous ACN containing 0.1% TFA (but no SDS) enhanced resolution and greatly increased column lifetime. Comparable solvents (with some variation in ACN and TFA concentrations) are also useful with agarose SE-HPLC columns [59]. Cereal protein SE-HPLC has been reviewed by Autran [58]. 

Many cereal endosperm storage proteins—especially glutelins— are, in their native forms, disulfide-bonded oligomers or polymers. Even if soluble, such proteins may resolve poorly upon RP-HPLC and elute as broad peaks [22], in part due to their many possible polymeric forms. Reduction is necessary to release disulfide-bonded subunits from cereal glutelins or oligomeric prolamins for analysis [19,32,42,76]. Thus, protein resolution, extractability, and stability are often improved by extraction under reducing conditions. To prevent reoxidation and further enhance resolution, resulting cysteine residues may be stabilized by alkylation, usually with 4-vinylpyridine [19,21,29,70,76]. 

---