Proteomic Analysis of the Blood of <i>Alligator mississippiensis</i>

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 <i>de novo</i> 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 <i>de novo</i> 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 <i>de novo</i> 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 <i>de novo</i> 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.

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

Chemistry

Year dc:date.available

2011

Author dc:creator

• Darville-Bowleg, Lancia Nadinia Fallen

• Lectin
• Antimicrobial Peptides
• Alligator mississippiensis
• Proteomics
• Mass spectrometry

Statement dc:rights

• withheld
• Secure the entire work for patent and/or proprietary purposes for a period of one year. Student has submitted appropriate documentation which states: During this period the copyright owner also agrees not to exercise her/his ownership rights, including public use in works, without prior authorization from LSU. At the end of the one year period, either we or LSU may request an automatic extension for one additional year. At the end of the one year secure period (or its extension, if such is requested), the work will be released for access worldwide.

Identifier

etd-10272011-153327
https://repository.lsu.edu/gradschool_dissertations/694

OAI identifier oai:identifier

oai:repository.lsu.edu:gradschool_dissertations-1693