Isoelectric focusing: Sample pretreatment – separation – hyphenation

Abstract

The three major goals of the research presented in this thesis are: • Investigation of electrophoretic sample pretreatment strategies. • Study and optimization of capillary isoelectric focusing for separation of proteins. • Hyphenation of isoelectric focusing to MS and iSPR. Chapter 2 gives an overview of the developments in cIEF over the past years including method improvements, new theoretical insights and applications. Chapter 3 describes the effect of several BGE additives on cIEF-UV repeatability and linearity and on protein signal intensity in MALDI-TOF MS. Chapter 4 describes the hyphenation of cIEF to MALDI-TOF MS via a spotting device. The development of the system as well as the effects of focusing and spotting times on separation efficiency are shown. A 3D plot is constructed from cIEF and MS data and the applicability is demonstrated for a degraded biopharmaceutical compound (glucagon). Chapter 5 is dedicated to the coupling of gel-IEF to iSPR via pressure blotting. The development of the system as well as the influence of the biological matrix on IEF separation and transfer efficiency is discussed. The applicability is demonstrated by the analysis of synovial fluid spiked with a known RA biomarker, i.e. citrullinated fibrinogen. Chapter 6 describes the use of the IonChip for salt removal from a protein sample prior to CZE separation (ITP-CZE mode) and the removal of interfering proteins from a peptide sample with subsequent CZE separation (CZE-CZE mode). Chapter 7 gives a general conclusion and recommendations for future research. Adjustments to the different systems and devices are proposed.