Characterization and Quantification of Covalent Modification of Proteins Using Mass Spectrometry

Identification and characterization of various post-translational modifications of protein is a key to understanding many unknown cellular processes. In the last few decades, mass spectrometry has evolved as an essential and effective analytical tool for qualitative and quantitative analysis of proteins. In this research, we have developed a novel MALDI-MS2 based quantification method for Desmosine and Isodesmosine, which served as cross-linking amino acids of elastin, in order to measure the elastin degradation in the body. This is the first quantification method that not only illustrates the potential of MALDI-Ion Trap MS2, but also improvement over the current LC-MS method, in terms of analysis time and solvent consumption, while maintaining similar analytical characteristics. The method is utilized to evaluate the time-dependent degradation of Des upon UV radiation (254nm) and result found to be consistent with quantification by 1H NMR. This work also involves the investigation of potential phosphorylation sites and evaluation of its role in various biochemical processes during HIV infection. Based on the results from different phosphorylation prediction algorithms, many in-vitro kinase assays were performed on HIV-derived peptides/proteins in presence of potential kinases. We have successfully identified few novel interactions between host-kinases/HIV phosphorylation substrates. These include the interactions of phosphorylation sites of Vif, Nef and Capsid proteins with protein kinase C (PKC), protein kinase A (PKA), and p38 MAPK respectively. Moreover, this work includes the development of cell-active inhibitors for cysteine cathepsins, a class of enzymes involve in many important cellular processes and in various disorders. In this study, we have synthesized library of two different classes of molecules containing oxirane and vinylsulfonate moieties. Various cell-based experiments were conducted to successfully demonstrate intracellular inhibition of cysteine cathepsin by these developed inhibitory molecules. The result of our study shows 2-(2-ehtylphenylsulfonyl) oxirane is cell-permeable and irreversible inhibitor of cathepsin B. On the other hand, peptidyl vinylsulfonate inhibitor (KD-1) is highly potent and selective cathepsin L inhibitor

Ultraviolet radiation reduces desmosine cross-links in elastin

Elastic fibers, a major component of the extracellular matrix of the skin, are often exposed to ultraviolet (UV) radiation throughout mammalian life. We report on an in vitro study of the alterations in bovine nuchal ligament elastic fibers resulting from continuous UV-A exposure by the use of transmission electron microscopy (TEM), histology, mass spectrometry, and solid state 13C NMR methodologies. TEM images reveal distinct cracks in elastic fibers as a result of UV-A irradiation and histological measurements show a disruption in the regular array of elastic fibers present in unirradiated samples; elastic fibers appear shorter, highly fragmented, and thinner after UV-A treatment. Magic angle spinning 13C NMR was applied to investigate possible secondary structural changes or dynamics in the irradiated samples; our spectra reveal no differences between UV-A irradiated and non-irradiated samples. Lastly, MALDI mass spectrometry indicates that the concentration of desmosine, which forms cross-links in elastin, is observed to decrease by 11 % following 9 days of continuous UV-A irradiation, in comparison to unirradiated samples. These alterations presumably play a significant role in the loss of elasticity observed in UV exposed skin