MALDI imaging allows the creation of a molecular image of a tissue slice. This image is reconstructed from the ion abundances in spectra that are obtained while rastering the laser over the tissue. These images can then be correlated with tissue histology to detect potential biomarkers of, for example, aberrant cell types. MALDI is known to have problems with ion suppression, making it difficult to correlate measured ion abundance with concentration. It would be advantageous to have a method that can provide more accurate protein concentration measurements, particularly for screening applications or for precise comparisons between samples. My hypothesis was that a method based on multiple reaction monitoring (MRM) with isotopically-labelled internal standards can be developed which would allow the accurate quantitation of proteins in MALDI Imaging. This study reports on the development of this novel MALDI Imaging method for the localization and accurate quantitation of proteins in tissues. This method involves optimization of in-situ tryptic digestion, followed by reproducible and uniform deposition of an isotopically-labelled standard peptide from a target protein onto the tissue, using an aerosol-generating device. Data is acquired by MALDI-MRM-MS and accurate peptide quantitation is determined from the ratio of MRM transitions for the endogenous unlabelled proteolytic peptides to the corresponding transitions from the applied isotopically-labelled standard peptides. In a parallel experiment, the quantity of the labelled peptide applied to the tissue was determined using a standard curve generated from MALDI-TOF-MS data. This external calibration curve was then used to extrapolate the quantity of endogenous peptide in a given area. All standard curves generated by this method had coefficients of determination greater than 0.97. These proof-of-concept experiments using MALDI MRM-based imaging show the feasibility of obtaining precise and accurate quantitation of tissue protein concentrations over two orders of magnitude, while maintaining the spatial localization information for the proteins. / Graduate

Correct number and pattern of digits is determined in a time and concentration-dependent manner by a gradient of Sonic Hedgehog (SHH) across the anterior-posterior axis of the embryonic limb bud. Owing to the potent morphogenic/mitogenic capabilities of SHH, transcription of the SHH gene in the limb is tightly regulated by feedback loops with other signalling pathways and by the Zone of Polarising Activity regulatory sequence (ZRS). The ZRS is a long-range, cis-regulatory limb-specific enhancer of SHH, and is essential for correct limb SHH expression. The Silkie, a polydactylous breed of chicken, possesses a C > A mutation in the ZRS, resulting in ectopic SHH expression in the anterior limb and hindlimb-specific polydactyly. We employ the Silkie mutant to investigate how SHH is regulated by the ZRS, and how Hedgehog signalling can modulate SHH expression in an autoregulatory manner. We further characterise the effects that the Silkie mutation has on subsequent limb development; investigating the dependence of increased posterior SHH, increased Hedgehog-dependent growth and necessary genotype in both the posterior and anterior limb bud. Several fundamental questions regarding SHH during limb development have yet to be fully addressed: how much SHH protein is present, and does it form a gradient as hypothesised by Wolpert’s Morphogen Gradient Model? By developing a standard curve-based method to assess absolute quantities of processed SHH protein, N-SHH, we find that the quantity of N-SHH protein increases through limb development, and does indeed form a quantifiable gradient across the posterior limb. By comparing quantity of N-SHH protein in equivalently staged mouse, rat, emu and chicken limbs, we find that there is no significant link between N-SHH protein quantity and digit number between mammalian and avian species, and investigate how digit number is modulated in the late limb. A number of species exhibit reduced numbers of digits, including the wings of the emu, cassowary and kiwi. Unlike in mammalian examples of digit loss (i.e. cow, pig) the emu wing has delayed and significantly reduced SHH expression. Through sequencing and functional in vivo testing of ZRS sequences of ratite bird species, we investigate whether the ZRS has a role in evolutionary digit loss. We also demonstrate the aspects of digit loss and Hh signalling are shared with examples of mammalian digit loss. This thesis presents novel research into multiple aspects of genetic regulation, limb development, and evolutionary developmental biology; elucidating both long held dogmas and upcoming areas of limb development.

In the last two decades, protein drugs have enjoyed a rapid growth and achieved a tremendous success in treating human diseases. However, the presence of physiological barriers greatly impedes the efficient delivery of such unconventional large molecule drugs, and therefore limits their clinical utility. An elegant way to address this challenge takes advantage of certain endogenous transporter proteins, such as human transferrin (Tf), whose ability to traverse physiological barriers has been extensively exploited. However, methods to investigate Tf-based drug delivery remained insufficient and unsatisfactory until recent development of quantitative mass spectrometry (MS). Hereby, MS-based methods have been developed and validated for quantitation of exogenous Tf in biological fluids. Particularly, different O18-labeling based approaches have been evaluated, modified and developed in this work, in order to achieve the most reliable quantitation. Alternatively, a novel approach based on indium labeling and inductively coupled plasma mass spectrometry (ICP-MS) detection has been developed for sensitive quantitation of Tf in biological fluids. The second aspect of this dissertation work focuses on the application of MS-based methods for characterization of protein drugs at different levels, ranging from protein identification, covalent structure, conformation, and interaction with physiological partners. Particularly, an O18-labeling assisted approach has been developed to identification of protein deamidation products. This new approach can readily distinguish between the two deamidated isomers. Also, an LC-MS based method has been developed for ranking the susceptibility of protein disulfide bonds to reduction, which could be applied to several disulfide bond-related analyses. Finally, a recently designed growth hormone transferrin fusion protein was studied using MS-based methods, and the molecular basis for its successful oral delivery was revealed.