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

Punprabhashi Vidanapathirana, Louisiana State University
Farhana Hasan, Louisiana State University
Kaitlyn Mussio, Louisiana State University
Anuja Pande, Louisiana State University
Michael Brands, Louisiana State University
Noureen Siraj, Louisiana State University
Anne Grove, Louisiana State University
Isiah M. Warner, Louisiana State University


© 2017 Elsevier B.V. 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, 66.5 kDa), ovalbumin (Ova, pI-4.6, 44.3 kDa), α-chymotrypsinogen (α-Chy, pI-8.8, 25.7 kDa), myoglobin (Myo, pI-6.8, 16.9 kDa), and cytochrome c (Cyt c, pI-10.0, 12.3 kDa). 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.