Proteomics - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 416

small peptide tracers, as the number of receptors per cell is small; the peptide tracer
must be highly receptor specific and the radiolabel should be free from its reactivity
toward peptide receptor-binding affinity. Apart from that, RRAs share some common
problems encountered in bioassays, particularly those pertinent to variations within
and among cell receptor preparations and the production of artifacts during incuba-
tion; even the presence of partial agonists or antagonists will result in unpredictable
changes in the final outcome.
Chromatography consists of a plethora of separation and resolution techniques
based on affinities of a solute for mobile and stationary phases. In the past, tech-
niques like thin layer chromatography have been widely explored. Currently, HPLC
has gained immense popularity for resolution and purification of proteins and pep-
tides. The reason for HPLC being so popular is because of technical flexibilities
and improvements in the preparation of inert, pressure-resistant, stationary phase
materials. This technique offers some unique advantages such as simplicity, speed,
and versatility, and is validated for pharmaceuticals and variety of biomolecules.
The modifications made in current HPLC are broadly categorized into three types:
(1) reversed phase, (2) ion exchange, and (3) gel permeation or size exclusion.
8.7.3.3 Reversed-Phase HPLC
Reversed-phase HPLC (RP-HPLC) is more applicable to analysis and the prepara-
tory stage for peptides. This technique differentiates peptides on the basis of differ-
ences in their hydrophobicity. In reverse phase chromatography, since the mobile
phase used is polar in nature, column retention is largely dependent upon the affi-
nity of hydrophobic segment of a peptide for non polar stationary phase as well as
the former's interaction with non polar components in eluting phase, if any. Meek
and Rosetti [41] have investigated factors affecting RP-HPLC retention and resolu-
tion of small peptides (i.e., those containing 20 or fewer residues). Results from their
experiments depict the role of amino acid hydrophobicity in retention of substance
or analyte in the column. Amino acids with aromatic or aliphatic side chains increase
retention time due to their hydrophobicity, whereas those with polar side chains are
either neutral or produce very little negative effect on retention time. Some practi-
cally observed deviations from the theoretically calculated retention times are mainly
due to reactivity of free silanol groups that undergo interaction with cationic solutes,
resulting in increased noise level or band broadening. For smaller peptides, there is
a decrease in retention time in the reversed-phase; silica generally decreases with
increasing volume of organic solvent in the mobile phase. Separation of very hydro-
phobic and/or larger peptides may require mobile phases of higher organic solvent
content. Determination of peptide homogeneity is one of the principal applications of
RP-HPLC, particularly in synthetic peptides, because chemical reactions can result
in altered biological activity of the peptide. Ongoing active research is focused on
determining the in vivo stability of proteins. However, current methodologies have
failed to succeed due to inherent complications resulting in compromised sensitivity
and selectivity.