Capillary electrophoresis (CE) was coupled off-line with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the analysis of proteins and peptides. CE fractions were collected directly on a matrix-coated MALDI target, using a sheath-flow interface. Protein adsorption during CE separations was prevented by coating the capillaries with the physically adsorbed, cationic polymer PolyE-323. The CE/MALDI-MS system was used for the analysis of model proteins and peptides at physiological pH as well as analysis of proteins in tear fluid. Moreover, tryptic on-target digestion of the collected protein fractions, with subsequent MALDI-MS and MS/MS peptide analysis, was demonstrated.
The first part of the work describes a procedure of oligonucleotide purification using a reversed-phase cartridge. The developed method employs a very efficient yet mild oligonucleotide detritylation on the cartridge support allowing fast purification of oligonucleotides regardless of their 5´-modification. Thiol- and amino-modified oligonuc-leotides were detritylated and purified with the same high efficiency as non-modified oligonucleotides. The method enables fast, parallel and automated purification of many oligonucleotide probes that was not possible before. In combination with the method of removal of tritylated failure fragments oligonucleotides were produced with purity superior to that of oligonucleotides purified using RP HPLC.In the second part of the present study a method of solid-phase RNA synthesis using 2´-tert-butyldithiomethyl (2´-O-DTM) is discussed. The stability of the DTM group during oligonucleotide assembly and deprotection in ammonia, together with its ability for rapid deprotection under mild conditions, allowed the synthesis of RNA with the quality similar to that of synthetic DNA oligonucleotides. The advantage of the 2´-O-DTM group is that it is completely orthogonal to all protecting groups used for the traditional solid-phase DNA synthesis. Therefore, the synthesis can be performed using a standard DNA synthesis procedure – no changes are needed for the product assembly. RNA oligonucleotides synthesized with retained 5´-terminal trityl group can be subjected to a cartridge-based purification using the procedure described in the first part of the study. The phosphoramidite synthesis was optimized for a large scale preparation and gives versatility for introduction of other alkyldithiomethyl groups according to the preference to their certain properties.The third part of the thesis describes the synthesis of a dithiomethyl linker and its utility for reversible conjugation of oligonucleotides. A dithiomethyl group, cleavable under mild conditions, was introduced onto 3´-OH of tritylated nucleosides via 3´-O-methylthiomethyl derivatives. The influence of different alkyl substituents on the disulfide bond stability was investigated, and stable analogues were employed in oligosyntheses. Two applications were developed using the present linker: 1) purification of oligonucleotides linked to the solid support; and 2) cartridge-based purification of tritylated oligonucleotides having an additional hydrophobic group on their 3´- terminus.
The combination of capillary electrophoresis (CE) and mass spectrometry (MS) constitutes a powerful microanalytical system in the fields of biology, medicine and chemistry. This thesis describes the development of three novel capillary coatings and demonstrates how these extend the utility of CE as a high-efficiency separation technique in protein analysis and biopharmaceutical drug screening. Due to the rapidly growing interest in characterizing the human proteome, there is an increased need for rapid protein separations. The use of CE in protein analysis is, however, nontrivial due to problems with protein adsorption to the fused-silica capillary walls. In this thesis, this problem was addressed by developing two novel, physically adsorbed, cationic polymer surface coatings, denoted PolyE-323 and Q-agarose. By using simple rinsing protocols, highly reproducible coatings, stable over a wide range of pH 2-11 were generated. Successful protein separations using cationic-coated capillaries in CE-MS, equipped with either electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI), has been demonstrated. In the pharmaceutical industry, favorable pharmacokinetic properties of a candidate drug, such as high bioavailability after oral administration, are crucial for a high success rate in clinical development. Tools for prediction of biopharmaceutically relevant drug properties are important in order to identify and discard poor candidate drugs as soon as possible. In this thesis, a membrane mimetic coating was developed by electrostatically immobilizing liposomes to the capillary wall, via an anchoring sublayer of Q-agarose. The liposome-coated capillaries were demonstrated in on-line CE-MS for prediction of drug membrane permeability.
Abstract A procedure for enhanced capillary electrophoresis‐electrospray ionization‐mass spectrometry (CE‐ESI‐MS) of proteins is presented. The use of a newly presented capillary coating, PolyE‐323, provided fast separations of typically a few minutes with high efficiency, good deactivation, and no bleeding into the mass spectrometer. Capillaries coated with PolyE‐323 showed high stability over a range of pH 2–10, and tolerance towards methanol and acetonitrile, two modifiers commonly used in CE‐ESI‐MS. Due to the speed and simplicity of the coating procedure, the polymeric surface could, if necessary, easily be regenerated. This capability is especially valuable when working with samples of complex matrix, where a capillary surface cleaning step might be desired in order to eliminate possible memory effects. The potential of PolyE‐323‐coated capillaries in bioanalysis using CE‐ESI‐MS was demonstrated by analyzing peptides and proteins up to 66 kDa using time of flight (TOF)‐MS. Due to the stable, anodal electroosmotic flow generated by the coating, the use of a sheathless ESI interface was enabled, demonstrated in peptide analysis with attomole sensitivity. The fast on‐line CE‐ESI‐TOF system using PolyE‐323‐coated capillaries provided efficient separation and detection of a large number of peaks in a short time, exemplified by the analysis of a tryptic digest of bovine serum albumin (BSA). The capability of the developed capillary surface coating was demonstrated by the separation of human plasma and cerebrospinal fluid (CSF).
Various approaches for removal of high‐abundance components in body fluids are currently available. While most methods are constructed for plasma depletion, there is a need for body‐fluid‐specific strategies. The aim of the present study was to design an affinity matrix suitable for the depletion of high‐abundance proteins in CSF (cerebrospinal fluid). Hence, molecules with specific affinity towards proteins present at high concentration in CSF were desired. Affibody® molecules are specific binders of small size that have shown high stability under various conditions and are therefore good candidates for such a matrix. The protein composition in CSF resembles that in plasma. However, 20% of the proteins are brain‐derived and are therefore present in higher proportions in CSF than in plasma, whereas larger plasma‐derived proteins are less abundant in CSF. Therefore five high‐abundance CSF proteins were chosen for the design of a CSF‐specific depletion setup. Affibody® molecules with specificity towards HSA (human serum albumin), IgG, transferrin and transthyretin were combined in an affinity column. In addition, polyclonal antibodies against cystatin C were coupled to chromatographic beads and packed in a separate column. Highly reproducible and efficient removal of the five target proteins was observed. The proportion of depleted proteins were estimated to be 99, 95, 74, 92 and 83% for HSA, IgG, transferrin, transthyretin and cystatin C respectively. SDS/PAGE analysis was used for monitoring and identifying proteins in native CSF, depleted CSF samples and the captured fractions. Moreover, shotgun proteomics was used for protein identification in native as well as depleted CSF and the achieved data were compared. Enhanced identification of lower‐abundance components was observed in the depleted fraction, in terms of more detected peptides per protein.
A bottom-up proteomic approach, based on capillary electrophoresis (CE), in combination with matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-ToF/ToF) mass spectrometry, was used to analyze immunoaffinity depleted human cerebrospinal fluid (CSF) and compare it with a non-depleted sample. After enzymatic digestion and desalting, the tryptic peptides were separated by CE using PolyE-323 modified capillaries and fractionated off-line onto MALDI target plates for further analysis by MALDI and tandem mass spectrometry (MS/MS). The protein profile of the depleted sample was compared with non-depleted CSF. Overall, 84 proteins were identified with 95% significance in both samples. The significance scores for the proposed biomarkers, such as amyloid-like protein 1 precursor, could be increased up to 12 times after the depletion. Other proteins, often suggested to be related to neurodegenerative diseases, such as amyloid beta A4 protein precursor, superoxide dismutase and apolipoprotein E precursor could only be found in the depleted CSF samples. The effect of a derivatization of tryptic peptides with 2-methoxy-4,5-dihydro-1 H-imidazole reagent for protein identification by mass spectrometry was also employed to increase the number of identified proteins and the sequence coverages. The results presented in this study illustrate the benefit of combining a sample pre-fractionation step and a label's ability to enhance the ionization efficiency with the potential of CE using PolyE-323 modified capillaries in the analysis of complex samples. The straight-forward approach that provides speed and simplicity resulting in high-resolution separations and low sample consumption represents an easily applicable separation technique that can serve as a complement to other currently existing analytical approaches needed in modern proteomic analysis of clinically relevant samples.
The increased knowledge about the complexity of the physiological processes increases the demand on the analytical techniques employed to explore them. A comprehensive analysis of the entire sample ...
