Separation of Polypeptides
by Isoelectric Point Focusing
in Electrospray-Friendly Solution Using a Multiple-Junction Capillary
Fractionator
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Abstract
We introduce an online multiple-junction capillary isoelectric
focusing fractionator (OMJ-CIEF) for separation of biological molecules
in solution by pI. In OMJ-CIEF, the separation capillary is divided
into seven equal sections joined with each other via tubular Nafion
membrane insertions. Each junction is communicated with its own external
electrolytic buffer which is used both to supply electrical contact
and for solvent exchange. The performance of the fractionator was
explored using protein and peptide samples covering broad pI range.
Separation was achieved in ionic and ampholytic buffers, including
ammonium formate, ammonium hydroxide, histidine, and arginine. By
maintaining electric potential across upstream segments of the capillary
after the focusing stage, selective release of downstream analyte
fractions could be achieved. The selective release mode circumvents
the problem of peak broadening during mobilization and enables convenient
comprehensive sampling for orthogonal separation methods. Using single-component
ampholyte buffers with well-defined pI cutoff values, controlled separation
of protein mixture into basic and acidic fractions was demonstrated.
The device is cheap and easy to fabricate in-house, simple in operation,
and straightforward in interfacing to hyphened analytical platforms.
OMJ-CIEF has a potential of becoming a practical add-on unit in a
wide range of bioanalytical setups, in particular as a first-dimension
separation in mass spectrometry based proteomics or as a preparative
tool for analyte purification, fractionation, and preconcentration