Doctor of Philosophy (PhD)



Document Type



My research focus was to investigate alligator blood using mass spectrometry-based proteomics methods to understand their innate immune systems. The first goal was to sequence peptides and proteins from the blood serum and leukocytes using tandem mass spectrometry and de novo sequencing. The second goal was to determine the function of these biological molecules and their relationship to the immune system. One- and two-dimensional gel electrophoresis was used to separate proteins from alligator leukocytes, which were enzymatically digested. The peptides were measured using reversed phase nano-high performance liquid chromatography coupled with tandem mass spectrometry (nano-HPLC-MS/MS) followed with de novo sequencing. The results, as described in Chapter 3 show that alligator leukocytes contain proteins that are similar to proteins found in other vertebrates such as mammals and reptiles that are related to immune responses. Isolation of small molecule interferences and peptides exhibiting antimicrobial activity from alligator leukocyte extracts are described in Chapters 4 and 5. Reversed-phase HPLC was used to separate the leukocyte mixture and antimicrobial activity tests were used to determine the active fractions. Interferants, EDTA and spermine were present and showed activity in early fractions. Two major peptides measured at 4.7 and 4.9 kDa in an active fraction were further separated on the basis of their charge, size and shape using ion mobility-mass spectrometry (IM-MS). Due to the limited fragmentation of the peptides using IM-MS, the peptides were isolated and fragmented using MALDI TOF/TOF MS for de novo sequencing. Lectins are a class of carbohydrate selective proteins that are part of the complement immune system. Chapter 6 presents results for a lectin isolated from alligators that have mannan and mannose binding activity. In this study, the monomeric lectin was isolated and enzymatically digested using five different proteases to create small and large peptides which were analyzed by LC-MS/MS. The peptides were determined via de novo sequencing and overlapped to generate the lectin sequence. Lectins may have varying degrees of glycosylation, therefore deglycosylation procedures suitable for mass spectrometry analysis are described in Chapter 7. Conclusions and future directions for the work in this dissertation will be summarized in Chapter 8.



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Committee Chair

Murray, Kermit K.



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Chemistry Commons