Presently, progress in the field of phosphoproteomics has been accelerated by mass spectrometry. This is not a surprise owing to not only the accuracy, precision and high-throughput capabilities of MS but also due to the support it receives from informaticians whom allow the automated analysis; making the task of going from a complex sample to a statistically satisfactory set of phosphopeptides and corresponding site positions with relative ease. However, the process of identifying and subsequently pinpointing the phosphorylation moiety is not straightforward and remains a challenging task. Furthermore, it has been suggested that not all phosphorylation sites are of equal functional importance, to the extent that some may even lack function altogether. Clearly, such sites will confound the efforts towards functional characterisation. The work in this thesis is aimed at these two issues; accurate site localisation and functional annotation. To address the first issue, I adopt a multi-tool approach for identification and site localisation; utilising the different underlying algorithms of each tool and thereby allowing an orthogonal perspective on the same tandem mass spectra. Doing so enhanced accuracy over any single tool by itself. The power of this multi-tool approach stemmed from its ability to not predict more true positives but rather by removal of false positives. For the second issue, I first investigated the hypothesis that those of functional consequence exhibit stronger phosphorylation-characteristic features such as the degree of conservation and disorder. Indeed, it was found that some features were enriched for the functional group. More surprisingly, there were also some that were enriched for the less-functional; suggesting their incorporation into a prediction algorithm would hinder functional prediction. With this in mind, I train and optimise several machine-learning algorithms, using different combinations of features in an attempt to (separately) improve general phosphorylation and functional prediction.

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Background - The role of protein phosphatases (PPs) during ischemic preconditioning in the rabbit heart was examined. Methods and Results - Fostriecin, a potent inhibitor of PP2A, was administered to isolated rabbit hearts starting either 15 minutes before or 10 minutes after the onset of a 30-minute period of regional ischemia and continuing until the onset of reperfusion. After 2 hours of reperfusion, infarct size was measured with triphenyltetrazolium chloride. In a second study with isolated rabbit cardiomyocytes, the effect of fostriecin pretreatment was assessed by measuring changes in cell osmotic fragility during simulated ischemia. PP1 and PP2A activities of isolated control and ischemically preconditioned cells were also measured. In a third series of experiments, left ventricular biopsies of isolated rabbit hearts were obtained before and at selected times during 60 minutes of global ischemia, and the tissue was assayed for PP1 and PP2A activities. In isolated hearts pretreated with fostriecin, only 8% of the ischemic zone infarcted, significantly less than that in untreated control hearts (33%; P<0.001) but comparable to that in ischemically preconditioned hearts (9%; P<0.001 versus control). Significant protection was also observed in the hearts treated only after the onset of ischemia (18% infarction; P<0.05 versus control). In isolated myocytes, fostriecin also provided protection comparable to that produced by metabolic preconditioning. Preconditioning had no apparent effect on the activity of either PP1 or PP2A in isolated ventricular myocytes or ventricular tissue obtained from heart biopsies. Conclusions - Fostriecin, a potent inhibitor of PP2A, can protect the rabbit heart from infarction even when administered after the onset of ischemia. But inhibition of either PP1 or PP2A does not appear to be the mechanism of protection from ischemic preconditioning.

Tyrosine phosphorylation is an important post-translational modification in cells that modulates key cellular pathways. Tyrosine phosphatases are the class of enzymes that remove this modification from proteins, yet we know relatively little about how they are regulated in various signaling contexts. Activity-based probes that successfully target active sites of tyrosine phosphatases and report on their activities can fill in this gap. We show the assessment of various thiol-reactive groups for their ability to target catalytic cysteine residues with specificity. Then we construct and screen a library of fragment-like compounds for their on-target and off-target reactivities. We also discuss theoretical considerations for screening covalent inhibitors for their kinetic parameters and show this using our experimental data. Lastly, we augment compounds selected from the library to enable click chemistry for reporter group attachment for use on the whole proteome, ultimately through mass spectrometry-based proteomics methods. We show enrichment of target proteins. These target-centric design efforts will yield new insights into the general development processes of activity-based probes or inhibitors.

Mammalian sperm are not able to fertilize immediately upon ejaculation; they become fertilization-competent after undergoing changes in the female reproductive tract collectively termed capacitation. Although it has been established that capacitation is associated with an increase in tyrosine phosphorylation, little is known about the role of this event in sperm function. In this work we used a combination of two dimensional gel electrophoresis and mass spectrometry to identify proteins that undergo tyrosine phosphorylation during capacitation. Some of the identified proteins are the mouse orthologues of human sperm proteins known to undergo tyrosine phosphorylation. Among them we identified VDAC, tubulin, PDH E1 β chain, glutathione S-transferase, NADH dehydrogenase (ubiquinone) Fe-S protein 6, acrosin binding protein precursor (sp32), proteasome subunit alpha type 6b and cytochrome b-c1 complex. In addition to previously described proteins, we identified two testis-specific aldolases as substrates for tyrosine phosphorylation. Genomic and EST analyses suggest that these aldolases are retroposons expressed exclusively in the testis, as has been reported elsewhere. Because of the importance of glycolysis for sperm function, we hypothesize that tyrosine phosphorylation of these proteins can play a role in the regulation of glycolysis during capacitation. However, neither the Km nor the Vmax of aldolase changed as a function of capacitation when its enzymatic activity was assayed in vitro, suggesting other levels of regulation for aldolase function. Looking upstream the kinase cascade, the identity of the kinase (s) that brings about the phosphorylation of the tyrosine residues remains to be elucidated. It has been suggested that the non receptor tyrosine kinase Src family is involved in the capacitation associated phosphorylation cascade. Using an immunological approach we show that the only Src family member present in mouse sperm extract is Src. The capacitation associated tyrosine phosphorylation is greatly reduced in the presence of Src specific inhibitors (SU6656 and SKI606) in vivo. As a means of control for the activity of Src inhibitors in our system, parallel experiments assaying the activity of PKA both in vivo and in vitro were realized. Surprisingly, Src inhibitors down regulates the phosphorylation of serine/threonine residues that correlate on earlier events in the capacitation, as assayed by western blot with PKA substrates antibody. However, in vitro kinase activity of PKA showed no effect of Src inhibitors in the phosphorylation of the PKA specific substrate, kemptide.

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The 55K product of early region 1B (E1B) of human adenoviruses is required for viral replication and participates in cell transformation. Both biochemical and genetic approaches have been used to show that this 496-residue (496R) protein of adenovirus (Ad5) is phosphorylated at both serine and threonine residues at sites near the carboxy terminus within sequences characteristic of a casein kinase II. Mutations which converted serines 490 and 491 to alanine residues decreased virus replication and reduced the efficiency of transformation of primary baby rat kidney cells, suggesting that phosphorylation at these sites is of importance in the regulation of 55K biological activity. / Thesis / Master of Science (MS)