Hyphenation of 2D heart cutting CIEF/CE-MS

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

Marburg

Workshop; CE FORUM 2014; 2014

The hyphenation of capillary isoelectric focusing (CIEF) with mass spectrometry is a very promising tool for 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 glycoprotein isoforms is still unsolved and 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, 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 CE dimension. A microfluidic chip device with a common intersection for both separation dimensions is used as the interface for hyphenation. This intersection defines the injection volume for the second dimension. Two aspects of method development are presented here: 1) CIEF/CE-MS sampling experiments: First 2D experiments show that the pH gradient can sequentially be heart cut and injected via the microfluidic chip interface into the second CE separation dimension and the proteins of interest can finally be detected via MS detection. This is not possible for the direct coupling of CIEF to MS detection because of analyte ion suppression due to the used ampholytes. We here show the principal application for myoglobin as a model analyte. 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. Depending on the stability of the used coating the anolyte and catholyte can be used to prevent the pH gradient to build up behind the detection window. We here demonstrate that the combination of 20 mM phosphoric acid as anolyte and 250 mM triethylamine as catholyte itself is suitable to spatially control the CIEF run. Numerical modeling is applied in order to support the experimental work, allowing the optimization of the relative concentrations of ampholytes and spacers.