Capillary isoelectric focusing for the characterization of glycoproteins

SYDES, DANIEL; KLER, PABLO A.; HUHN, CAROLIN

Essen (NRW)

Congreso; ANAKON 2013; 2013

GDCH

The hyphenation of capillary isoelectric focussing with mass spectrometry (cIEF-MS) is a very promising tool regarding the characterisation of proteins and peptides, particularly for R&D and quality control of monoclonal antibodies[1]. However, the coupling between cIEF and MS is still challenging. The high number of carrier ampholytes (Cas) screen the MS signal inhibiting the proper MS detection of analytes. The unambiguous identification of glycoprotein isoforms is still unsoveld, and currently mostly involves off-line sample preparation steps. Several attempts have been made in order to remove the ampholytes prior to enter the MS: The use of a reversed-phase liquid chromatography (RPLC), dialysis interfaces, and complex microfluidic devices are mentioned in the literature[2-4]. In our work we seek to establish a 2D separation approach via cIEF/CE-MS to separate analytes from carrier ampholytes, which are not compatible with MS, in the second CE separation column. We here show our initial results for the establishment of a hybrid multicapillary-microchip 2D separation for cIEF/CE-MS with a focus on the optimization of the cIEF separation regarding its compatibility with subsequent CE separation. To inhibit adsorption of proteins to the capillary surface, and to minimize the electroosmotic flow, different coating strategies were applied[5]: We focused on neutral adsorbed coatings to avoid labor-intensive covalently coupled coatings. Also, optimization of the mobilization process with a focus on the mobilization from the inlet side to transfer fractions of the cIEF into the CE capillary was performed. Pressure and chemical mobilization procedures are compared. Furthermore tools were developed to detect and control the position of the IEF stack at the outlet side using iminodiacetic acid and tetramethylethylenediamine (TEMED) in order to facilitate sample transfer to the second column. Here we show the results of the work with standard proteins (pI markers, bovine serum albumin, glycosylated fetuin, RNAse A and glycosylated RNAse B) using PharmalyteTM ampholyte mixtures covering the respective pI ranges of the proteins.