Proteins have such structural features that they may interact with different types of surfaces by all possible forces, i.e., electrostatic, hydrogen bonding, hydrophobic. In this thesis two different types of coatings for fused silica capillaries aimed to eliminate such interactions have been studied. The first is a covalent, electroosmosis-free coating with polyacrylamide (PAA) and the second involves a non-covalent coating with the quaternary ammonium compound N, N-didodecyl –N, N- dimethylammonium bromide (DDAB) with a strong anodic electroosmosis. Optimal conditions regarding efficiency and resolution were established by variations of the composition and ionic strengths of buffers at pH below the isoelectric point of the proteins. To achieve high efficiency and resolution the choice of buffer constituents was extremely important. The PAA coating was very stable at neutral and acidic conditions. Ammonium acetate (0.12 M) and ammonium hydroxyacetate (0.15 M) both at pH 4 provided the best separations with plate numbers up to 1 700 000 plate/m that is among the highest reported in the literature. Capillaries coated with DDAB were stable enough to, without recoating, permit consecutive separations of the proteins up to 9 hours (90 injections). High apparent efficiencies (over 1 million plates/m) were achieved with ammonium acetate (0.07 M), ammonium hydroxyacetate (0.08 M) and sodium phosphate (0.1 M) at pH 4. Zone broadening was studied by determination of the variance contributions from all main parameters. Significant variances were contributions from longitudinal diffusion, capillary curvature, injection plug, detector time response and detector slit width while other variances, e.g., variances for Joule heat and vertical sedimentation were negligible. The remaining undetermined variance may have its origin in all types of relatively slow interactions including adsorption onto the capillary surfaces and protein-buffer component interactions. The results indicate that the latter is the main cause to zone broadening in protein separations.

Metabolism is the sum of all chemical processes with the purpose to maintain life, as well as enable reproduction, in a living organism. Through the study of metabolism, increased understanding of pharmacological mechanisms and diseases can be achieved. This thesis describes several ways of doing so, including targeted analysis of selected metabolites and investigations of systematic metabolic differences between selected groups through pattern recognition. A method for exploring metabolic patterns in urine samples after intake of coffee or tea was developed. The methodology was later used with the aim to find biomarkers for prostate cancer and urinary bladder cancer. Furthermore, a fully automated quantitative method was developed for concentration measurements of the double prodrug ximelagatran and its metabolites in pig liver. The method was then used to study the roll of active transporters in pig liver cells. Moreover, a fundamental study was conducted to investigate how monitoring of small, doubly charged analytes can improve the limit of detection and precision in a quantitative method. The techniques used for the experiments were liquid separation coupled to electrospray mass spectrometry. Extra efforts were made to make the separation and the ionization as compatible as possible to each other for increased quality of the collected data.

PEG-lipid stabilized bilayer disks have been found in lipid mixtures containing polyethylene glycol (PEG)-lipids where the combination of a high bending rigidity and low PEG-lipid/lipid miscibility favours disk formation. The disks are planar and circular in shape and their long-term stability is excellent. Theoretical calculations and experimental observations suggest that the micelle forming PEG-lipid are situated at the rim of the aggregate, protecting the hydrophobic lipid chains in the bulk of the aggregate from contact with water. This thesis deals with fundamental aspects concerning the lipid distribution in the disks, as well as with development, optimization, and initial evaluation of the disks as model membranes in partition and interaction studies. Small angle neutron scattering was used to study the partial segregation of components within the bilayer disk. The experiments verified that the PEG-lipids segregate and accumulate at the bilayer disk rim. The proof of component segregation is important from a fundamental point of view and useful, as exemplified in the below-mentioned study of melittin-lipid interaction, when interpreting partition or binding data obtained from studies based on bilayer disks. Today liposomes are often used as model membranes in partition and interaction studies. Using liposomes to predict, e.g., drug partitioning can however have certain drawbacks. In this thesis the disks were proven to be attractive alternatives to liposomes as model membranes in partition studies. The formation of bilayer disks by a technique based on detergent depletion enabled incorporation of a transmembrane protein in the bilayer disks and opened up for the use of disks as model membranes in membrane protein studies. Further, bilayer disks were used in a comparative study focused on the effect of aggregate curvature on the binding of the peptide melittin. Various techniques were used to perform initial evaluations of the bilayer disks as model membranes. Of these, capillary electrophoresis and biosensor-based technology had not been used before in combination with bilayer disks.

Different multiple-injection methodologies and the partial filling technique (PFT) have been utilized for separation of pharmaceuticals by capillary elec-trophoresis. In multiple-injection capillary zone electrophoresis (MICZE), the samples and all standards, used for construction of the calibration curve, are analyzed within a single run. Four different modes of MICZE have been described by means of equations, which were experimentally verified. The developed equations facilitate the transfer from conventional single-injection CZE to one or more of these MICZE-modes, depending on the selectivity between the analyte and the injection marker. The applicability of two of these modes was then demonstrated by quantification of buserelin and salbutamol, re-spectively in commercially available pharmaceutical products. The content of buserelin in an injection solution was determined to 0.94 mg/ml, which only deviated slightly from the declared concentration (1 mg/ml). An alter-native mode of MICZE, offering a higher number of sequential sample injec-tions, was then utilized for single-run determination of salbutamol in 15 tab-lets, with a labelled content of 8 mg. The average content of the tablets was determined to 7.8 mg, with an intra-tablet variation of 3 % or less. Moreover, UV- and mass-spectrometric detection of enantiomeric amines, resolved by non-aqueous capillary electrophoresis (NACE), was demon-strated. Separation of enantiomeric amines was achieved using the chiral selector (-)-2,3:4,6-di-O-isopropylidene-2-keto-L-gulonic acid, (-)-DIKGA. Introduction of the non-volatile (-)-DIKGA into the mass-spectrometer was avoided by using the PFT, where the capillary is only partially filled with electrolyte containing the chiral selector.

Performing quantitative analysis of targeted bioactive compounds in biological samples, such as blood plasma, cerebrospinal fluid or extracts from pig liver, put high demands on the ruggedness of the method acquiring the results. In addition to the complexity of the sample matrix, the bioactive compounds targeted for analysis usually have low levels of natural abundance, further increasing the demand on the analytical method sensitivity. Furthermore, quantitation of analytes at trace levels in the presence of large amounts of interfering species in biofluids must aim for repeatable precision, high accuracy ensuring the closeness to the true values, a linear response spanning over several orders of magnitude and low limits of quantitation to be valid for monitoring disease states in clinical analysis. An analytical method most commonly follow a certain order of events, called the analytical chain, which includes; experimental planning, sampling, sample pre-treatment, separation of species, detection, evaluation, interpretation and validation, all equally important for the outcome of the results. In this thesis, the scope has been to create novel methods, or to refine already existing methods, in order to achieve even better performances of the different events in the analytical chain. One of the aspects has been to sample and enrich analytes in vivo by the use of solid supported microdialysis, giving the advantage of almost real-time monitoring of analyte levels within a living host with targeted selectivity due to the analyte affinity for solid particles. Another aspect to selectively clean and enrich analytes in a complex matrix has been developed and automated on-line by the use of a column-switching technique before the analytical separation. By using several steps of extraction and separation coupled on-line to selected detection by the use of a triple quadrupole mass spectrometer facilitates great selectivity of species. The mass spectrometer also gives the ability to distinguish between isotopically labelled analogues coeluting with the analytes yielding the necessary accuracy for quantitative evaluation. Both development of analytical methods and clinical applications has been performed, as well as improvements of existing techniques, all to improve the quantitation of trace levels of targeted analytes in biofluids.

Two novel, single-isomer, sulfated cyclodextrins, the sodium salts of heptakis(2- O-methyl-3-O-acetyl-6-O-sulfo)cyclomaltoheptaose (HMAS) and heptakis(2-O-methyl- 6-O-sulfo)cyclomaltoheptaose (HMS) were used as chiral resolving agents in both aqueous and non-aqueous electrophoretic separation of a set of pharmaceutically active weak base enantiomers. Enantiomers of twenty one of the twenty four weak bases were baseline resolved in one or more of the background electrolytes (BGE’s) used. An eight-step synthetic method was used to produce, on a large scale, the title compounds in greater than 97% purity. The purity of the synthetic intermediates and the final products were characterized by HPLC-ELSD and indirect UV-detection capillary electrophoresis (CE), respectively. X-ray crystallography, MALDI-TOF mass spectrometry and 1H as well as 13C NMR spectroscopy allowed for unambiguous characterization of the structure of each intermediate and the final product.

No description available.

<p>Proteins are a highly diversified group of molecules, andfor their study, advanced analytical tools are required. Inparticular, a need for high-throughput techniques has emergedin order to enable the characterization of large sets ofproteins. In this thesis, improved techniques for proteinseparations as well as new tools for the mass spectrometricanalysis of proteins are described.</p><p>In the work, presented in the first part of the thesis, arefined extract containing proteases from Antarctic krill (<i>Euphausia superba</i>) was separated and characterized bymeans of capillary electrophoresis (CE) and mass spectrometry(MS). Tailored CE separations of the krill extract revealed thepresence of approximately 50 components. In addition, adetailed CE and MS analysis of fractions, containing individualkrill proteases has been carried out. Trypsin-like proteasesfrom krill exhibited a 12-fold and a 60-fold higher digestionefficiency at 37 °C and 2 °C respectively compared todigests performed with bovine trypsin. Furthermore, thecleavage specificity of the trypsin-like proteases wasstudied.</p><p>In the last part of the thesis, novel concepts forchip-based nanoelectrospray (nanoESI) and matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry are described.First, a micromachined silicon chip with a two-dimensionalmatrix of out-ofplane nanoESI needles for high-throughputanalysis was fabricated. A two-fold improvement insignal-to-noise reproducibility was obtained. Second, achip-based target for MALDI was developed, which featured pairsof elevated 50×50 µm anchors in close proximity. Theanchors were individually addressable with sample solution. Theminiaturized sample preparations at close distance to eachother allowed a simultaneous ionization of a physicallyseparated sample and standard by one single laser pulse. Thisresulted in a twofold reduction of relative mass errors.Moreover, ion suppression of the analyte was significantlyreduced. The effective utilization of the sample resulted in adetection limit of ca 200 zeptomole of angiotensin I.</p><p><b>Key words:</b>Proteins, peptides, proteases, Antarctickrill,<i>Euphausia superba</i>, capillary electrophoresis,fluorosurfactants, mass spectrometry, nanoelectrospray, ESI,MALDI, chip, high-throughput, reproducibility, sensitivity andmass accuracy.</p>

The analysis of large intact proteins and complex biological samples containing drug molecules is a common complicated task for many scientists. However, due to the importance of these molecules, there is a growing interest in pharmaceutical and medicinal research to develop rapid, highly sensitive and efficient analytical techniques. The advantages of capillary electrophoresis (CE) in combination with mass spectrometry (MS) provide a powerful analytical tool. However, further improvement and development of these techniques are required to extend their utility and to meet the challenges of selected analytes. Thus, the scope of this thesis deals with the development of novel analytical methods to achieve efficient and high performance analysis of peptides, intact proteins, digests of complex samples and basic pharmaceutical drug compounds in biological matrices. Implementation of CE for routine analysis of proteins and complex samples is constrained by the partial adsorption to the capillary wall. Consequently, the use of surface modified capillaries is required to control the surface properties and prevent analyte adsorption. In this thesis, analyte adsorption was successfully prevented using tailored covalent cationic (M7C4I) and electrostatic cationic (PVPy-Me) coatings. Rapid and efficient separations of peptides, proteins and digests of complex samples such as cerebrospinal fluids were obtained with these coatings. The M7C4I coating showed a distinct ability to handle large intact proteins with a molecular size of over 0.5 MDa. The highest peak efficiencies and surprisingly high peak stacking effects were obtained by adding salts to the protein samples. The effect of salt additives on peak efficiencies of intact proteins was further demonstrated and compared using different surface modified capillaries. Additionally, rapid CE-ESI-MS quantification of pharmaceutical drug molecules in human plasma was performed after a SCX-SPE sample preparation method using the M7C4I coating. In conclusion, the results presented in this thesis show the strong potential of CE in combination with MS using electrospray ionization (ESI) for the analysis of peptides and large intact proteins and the applicability for clinical monitoring of the levels of pharmaceutical drug molecules in human plasma with high sensitivity and efficiency. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 734

Advances in science and technology during the past decade have greatly enhanced the level of the structural investigation of macromolecules – peptides and proteins. Biological mass spectrometry has become one of the most precise and sensitive techniques in peptide and protein analysis. However, increasing demands of biotechnological applications require further progress to be made. In the present thesis the development and improvement of peptide and protein characterization methods and techniques based on ion-electron and ion-photon reactions in electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry are described. The focus is on the development of the electron capture dissociation method, recently discovered by the group of professor McLafferty, into a high rate, efficient tandem mass spectrometrical technique. The rate and reliability of the electron capture dissociation technique were greatly increased by implementation of low-energy pencil electron beam injection systems based on indirectly heated dispenser cathodes. Further implementation of a hollow electron beam injection system combined, in a single experimental configuration, two rapid fragmentation techniques, high rate electron capture dissociation and infrared multiphoton dissociation. Simultaneous and consecutive irradiations of trapped ions with electrons and photons extended the possibilities for ion activation/dissociation reaction schemes and lead to improved peptide and protein characterization. Using these improvements, high rate electron capture dissociation was employed in time-limited experiments, such as liquid chromatography–tandem mass spectrometry and capillary electrophoresis-tandem mass spectrometry. The analytical applications of the developed techniques have been demonstrated in top-down sequencing of peptides and proteins up to 29 kDa, improved sequencing of peptides with multiple disulfide bridges and secondary fragmentation (w-ion formation), as well as extended characterization of peptide mixtures separated by liquid chromatography and capillary electrophoresis. For instance, the dissociation of peptides resulting from enzymatic digestion of proteins provided complementary structural information on peptides and proteins, as well as their post-translational modifications.