Cationic ionic liquid surfactant-polyacrylamide gel electrophoresis for enhanced separation of acidic and basic proteins with single-step ribonuclease b glycoforms separation.

ABSTRACT

Cationic ionic liquids-based surfactants (ILS), such as 4-methyl pyridinium bromide (CnPBr, where n=4,6,8), were used in preparation of polyacrylamide gels, sample buffer, and running buffer for cationic ILS polyacrylamide gel electrophoresis (ILS-PAGE). These ILS are liquids in the pure state and were selected for improved separation of ribonuclease b (Rib b) glycoforms in a single step and a protein mixture containing bovine serum albumin (BSA, pI-4.8, 66.5kDa), ovalbumin (Ova, pI-4.6, 44.3kDa), α-chymotrypsinogen (α-Chy, pI-8.8, 25.7kDa), myoglobin (Myo, pI-6.8, 16.9kDa), and cytochrome c (Cyt c, pI-10.0, 12.3kDa). Results acquired for Rib b glycoform separation by use of ILS were compared with conventional non-ILS surfactants-PAGE sodium dodecylsulfate (SDS)-PAGE, cetyltrimethylammonium bromide (CTAB)-PAGE, and benzyldimethyl-n-hexadecylammonium chloride (16-BAC)-PAGE. A single protein band was observed with relatively short migration time in all the conventional PAGE techniques tested. In contrast, ILS-PAGE showed multiple bands with two distinct bands for Rib b protein. The two distinct bands of Rib b from ILS-PAGE were further analyzed using MALDI-MS. Examination of MALDI-MS spectral data revealed the presence of Rib b glycoforms. Furthermore, a two-dimensional isoelectric focusing (IEF)/SDS-PAGE map of Rib b protein revealed negative charge heterogeneity on the protein, which is a common observation for glycoproteins. This overall discovery greatly enhances the capability of using cationic ILS-PAGE for Rib b protein separation. Among all ILS tested, excellent protein separations were observed using C4PBr ILS at concentrations of 0.05% (w/v) in polyacrylamide gels, 0.01% (w/v) in protein sample buffer, and 0.1% (w/v) in running buffer. Under these optimum conditions, all other tested proteins were separated as sharp bands with good resolution.