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Binding of three ruthenium(ii) compounds of general formula mer-[Ru(L3)(N-N)X][Y] (where L3 = 4-chloro-2,2:6,2-terpyridine (Cl-tpy); N-N = 1,2-diaminoethane (en), 1,2-diaminocyclohexane (dach) or 2,2-bipyridine (bipy); X = Cl; Y = Cl) to human serum albumin (HSA) has been investigated by nano-LC/nano-ESI MS and docking studies. A bottom-up proteomics approach has been applied for the structural characterization of metallated proteins and the data were analyzed in both the positive and negative ion mode. The negative ion mode was achieved after the post-column addition of an isopropanol solution of formaldehyde that enabled sample ionization at micro-flow rates. The negative ion mode MS has been proved to be beneficial for the analysis of binding sites on ruthenated protein in terms of ion charge reduction and consequent simplification of target sequence identification based on isotopic differences between ruthenated and non-ruthenated peptides. Moreover, the negative ion mode ESI MS shows the advantage of singly charged ion formation and, unlike MALDI MS, it does not cause complete ligand fragmentation, merging the benefits of each method into a single experiment. Six target sequences were identified for the binding of en and dach compounds, and four sequences for the binding of bipy. All compounds have been found to bind histidine and one aspartate residue. Docking studies showed that the identified sequences are the constituents of five distinct binding sites for en and dach, or two sites for the bipy complex. The selection of binding sites seems to be dependent on the chelate ligand and the form of the complex prior or after hydrolysis of the leaving chloride ligand.

Neurosteroids are steroids synthesised in the central and peripheral nervous systems. Known neurosteroids include pregnenolone, dehydroepiandrosterone (DHEA), progesterone and its reduced metabolites. It has been demonstrated that neurosteroids modulate neurotransmission by binding to neurotransmitter receptors, and exert physiological functions that are clearly different from those of endocrine steroids. The effects of neurosteroids on improving the memory of cognitively impaired aged rats, on the inhibition of aggressiveness in castrated male mice, and trophic effects on neuronal regeneration and remyelination have been documented. The local synthesis, selective interaction with neurotransmitter receptors and behavioural effects of neurosteroids strongly suggests that they may have important physiological or pathophysiological roles. There is an increasing need to develop methods to analyse these hormones with high sensitivity and high specificity. In this thesis I focused on the development of methods combining nano-electrospray (ES) mass spectrometry with capillary column liquid chromatography (CLC) for the analysis of profiles of neurosteroids in rat brain. It was also an aim to make the methods applicable to a broad range of lipophilic biomolecules. Initially, synthetic steroid sulphates and unconjugated oxosteroids (ketosteroids) were studied by nano-ES and tandem mass spectrometry. Steroid sulphates could be detected as deprotonated molecules in full range scanned spectra at a level of 1 pg/µL. Information about steroid structure was obtained from collision-induced dissociation (CID) spectra of 1 ng of steroid sulphate, while characterisation of the sulphate ester group required only 3 pg of material. Unconjugated oxosteroids were converted into their oximes which were detected as protonated molecules with 20 times higher sensitivity than the underivatised steroids. The detection limits for the oximes of 3-oxo-delta4, 20-oxo and 17-oxo steroids were 2.5, 5

A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Furthermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS., QC 20100705

Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.

Purpose To compare the lipid composition of human meibum across three different meibum expression techniques. Methods Meibum was collected from five healthy non-contact lens wearers (aged 20-35 years) after cleaning the eyelid margin using three meibum expression methods: cotton buds (CB), meibomian gland evaluator (MGE) and meibomian gland forceps (MGF). Meibum was also collected using cotton buds without cleaning the eyelid margin (CBn). Lipids were analyzed by chip-based, nano-electrospray mass spectrometry (ESI-MS). Comparisons were made using linear mixed models. Results Tandem MS enabled identification and quantification of over 200 lipid species across ten lipid classes. There were significant differences between collection techniques in the relative quantities of polar lipids obtained (P<.05). The MGE method returned smaller polar lipid quantities than the CB approaches. No significant differences were found between techniques for nonpolar lipids. No significant differences were found between cleaned and non-cleaned eyelids for polar or nonpolar lipids. Conclusion Meibum expression technique influences the relative amount of phospholipids in the resulting sample. The highest amounts of phospholipids were detected with the CB approaches and the lowest with the MGE technique. Cleaning the eyelid margin prior to expression was not found to affect the lipid composition of the sample. This may be a consequence of the more forceful expression resulting in cell membrane contamination or higher risk of tear lipid contamination as a result of reflex tearing.© 2016 Elsevier Inc.

Purpose To compare the lipid composition of human meibum across three different meibum expression techniques. Methods Meibum was collected from five healthy non-contact lens wearers (aged 20-35 years) after cleaning the eyelid margin using three meibum expression methods: cotton buds (CB), meibomian gland evaluator (MGE) and meibomian gland forceps (MGF). Meibum was also collected using cotton buds without cleaning the eyelid margin (CBn). Lipids were analyzed by chip-based, nano-electrospray mass spectrometry (ESI-MS). Comparisons were made using linear mixed models. Results Tandem MS enabled identification and quantification of over 200 lipid species across ten lipid classes. There were significant differences between collection techniques in the relative quantities of polar lipids obtained (P<.05). The MGE method returned smaller polar lipid quantities than the CB approaches. No significant differences were found between techniques for nonpolar lipids. No significant differences were found between cleaned and non-cleaned eyelids for polar or nonpolar lipids. Conclusion Meibum expression technique influences the relative amount of phospholipids in the resulting sample. The highest amounts of phospholipids were detected with the CB approaches and the lowest with the MGE technique. Cleaning the eyelid margin prior to expression was not found to affect the lipid composition of the sample. This may be a consequence of the more forceful expression resulting in cell membrane contamination or higher risk of tear lipid contamination as a result of reflex tearing.

Dry eye is a highly prevalent disease causing symptoms of discomfort that are severe and disabling for many sufferers. One of the leading causes of the evaporative form of this complaint is believed to be Meibomian Gland Dysfunction (MGD). Although clinical manifestations of MGD are easily observed, the association between the morphology and function of the meibomian gland and the symptoms experienced by those affected, remains controversial. Better understanding of this relationship may help to predict those at risk and will contribute to development of effective treatments and preventative strategies.This thesis describes a series of studies designed to investigate the relationship between subjective ocular symptomatology and both meibomian gland morphology and function.Grading scales commonly used to assess meibomian gland morphology and lid wiper epitheliopathy were validated and found to offer only moderate sensitivity to detect changes. Objective assessment systems incorporating image analysis software were developed which permitted highly repeatable and accurate assessment of meibomian gland morphology and lid wiper epitheliopathy.The Korb meibomian gland evaluator and the meibomian gland forceps were identified as the optimal meibum expression methods.Tears and meibum were collected and analysed using nano-electrospray, mass spectrometry. These techniques were able to identify and quantify the major classes of component lipids. The lipid profiles derived showed good repeatability, both within and between days. A comparison between tears and meibum showed them to have similar lipid profiles, apart from phospholipids, which occurred roughly four orders of magnitude more abundantly in tears. This confirmed that meibum is not the major source of phospholipids in tears.No clear relationship between meibomian gland morphology and symptoms of discomfort was found, with the possible exception that meibomian gland coverage of the upper lid is reduced in those with mo

Mass spectrometry is an important analytical tool in biological and biochemical research. The speed, accuracy and sensitivity is unmatched by conventional analytical techniques. Identification of proteins and characterisation of their primary structure is a rapidly growing field in the post-genomic era, where matrix-assisted laser desorption/ionisation time-of-flight peptide mass fingerprinting combined with electrospray tandem mass spectrometry is well suited to solve the questions. This thesis deals with the strength and diversity of applying mass spectrometry to biochemical projects. The study covers sample preparation, protein identification, characterisation of post-translational modifications and mechanistic aspects of gas-phase cleavage of protonated peptides. The characteristics of a quadrupole - time-of-flight tandem mass spectrometer, used throughout this study, were investigated in a project where the thyroid hormone receptor ligand binding domain (TR-LBD) was analysed. Carboxymethylation of the TR-LBD in its folded conformation and identification of the alkylated Cys residues allowed estimates of tertiary structure relationships within the protein. In the tryptic digest, the peptides were mass measured with an accuracy of 2 ppm, and product ions generated by tandem mass spectrometry were measured to an accuracy of 20 ppm when internal calibrants were used. Proline-rich proteins (PRPs) are predominant in saliva and nano-electrospray mass spectrometry was employed to identify their degradation products after treatment with Streptococcus and Actinomyces microorganisms. Several of the peptides generated had a Cterminal Pro-Gln bacteria binding sequence, and release of a pentapeptide with innate immunity properties was suggested. In another study it was shown that PRP-1 and its Cterminally truncated form PRP-3 are present in two forms in human saliva, one of which is 0hexuronidated at Ser 17 in the biologically active N-terminal segment. Nano-electrospray tan