Dynamic coatings and cationic and anionic spacers for isoelectric focusing developments

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

Congreso; 20th INTERNATIONAL SYMPOSIUM ON ELECTRO- AND LIQUID PHASE- SEPARATION TECHNIQUES; 2013

The hyphenation of capillary isoelectric focusing (cIEF) with mass spectrometry (MS) is a very promising tool with respect to the characterization of proteins and peptides especially for R&D and quality control of monoclonal antibodies[1]. However, the coupling of cIEF with MS is still challenging due to the high number of carrier ampholytes being incompatible with MS detection. The unambiguous identification of e.g. glycoprotein isoforms is still unsolved and currently mostly involves off-line sample preparation steps. Several attempts have been made to remove the ampholytes prior to entering the MS: The use of a reversed-phase liquid chromatography (RPLC), dialysis interfaces as well as multidimensional microfluidic devices are mentioned in the literature[2-4]. We seek to establish a 2D separation approach for cIEF/CE-MS to separate analytes from carrier ampholytes in the second dimension CE separation column. Two aspects of method development are presented here: 1) Coatings: As we use a glass microchip as an interface for column coupling, the use of commercially available precoated capillaries is impossible. In order to also coat the microfluidic channels in the interface we focus on neutral or cationic adsorbed and covalently coupled coatings. We show that a combination of a covalently coupled polyvinyl alcohol coating together with celluloses and polyethylene oxide dynamic coatings provides very stable conditions and high repeatability, also when the microfluidic interface is included. With this combination, very sharp signals can also be obtained in the acidic pH-range. 2) Use of spacers: In order to be able to transfer IEF-focused signals of interest into the second dimension, the peak positions of the focused proteins have to be well controlled. Traditionally tetramethylethylene diamine (TEMED) is used to prevent the pH gradient to build up behind the detection window. We here demonstrate that the combination of TEMED and iminodiacetic acid is suitable to spatially control the cIEF run. Electrophoretic modeling is applied in order to support the experimental work, allowing optimizing the relative concentrations of ampholytes and spacers. [1] Z. Sosic et al., Electrophoresis 29 (2008) 29, 4368-4376. [2] F. Zhou et al., Anal. Chem. 76 (2004) 2734-2740. [3] D. Mohan et al., Anal. Chem. 75 (2003) 4432-4440. [4] Y.-C. Wang et al., Anal. Chem. 76 (2004) 4426-4431.