Author

Dan Wang

Date of Award

2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Clinical-Bioanalytical Chemistry

Department

Chemistry

First Advisor

Sun, Xue-Long

Subject Headings

Analytical Chemistry, Biochemistry, Chemistry, Immunology

Abstract

Sialic acids (SAs) are a diverse family of naturally occurring 2-keto-3-deoxy-nononic acids that are involved in a wide range of biological processes, including early fetal development, cellular recognition, and utilization by microbes. While it is clear that cell surface SAs are highly involved in the immune system, the sialylation status of individual immune cells and functions are still unknown. In this study, I combined the newly developed LC-MS/MS methods with flow cytometry and confocal microscopy to systematically study the sialylation and desialylation dynamics of macrophages at different conditions.

First, I developed an accurate LC-MS/MS method to quantify free SA in human plasma with isotope-labeled standard calibration and 3,4-diaminotoluene derivatization. This method is capable to distinguish SA analogus in complex biological samples, which paves the path for dynamic SAs research. Menwhile, another LC-MS/MS method with direct SAs quantification was developed for high throughtput analysis. This method does not require complicated sample preparation and can quantify SA at 2 ng/mL.

Next, I performed globally profiling of sialylation status of Raw 264.7 macrophages by flow cytometry, confocal microscopy, and LC-MS/MS. Both flow cytometry and confocal microscopy showed the predominat of a-2,3 linked SAs on the cell surface, and increase of a-2,6 linked SAs after atorvastatin treatment. Moreover, LC-MS/MS showed total SA increased 3 times upon treatment. Further experiment indicated the correlation of a-2,6 linked SAs with cell apoptosis.

Finally, I systematically examined the sialylation and desialylation profiles of THP-1 monocytes after differentiation and polarization. Both a-2,3 and a-2,6 linked SAs on the cell surface were decreased during diffrentiation, which was in accordance with the increased free SA in the medium and elevated activity of NEU1 sialidase. Meanwhile, the increase of SA expression during differentiation was evidenced by siaoglycoconjugates inside the cells and total SA in the cell lysate.

Overall, the combined approach has bee successfully applied to profile SAs in the cell culture system. LC-MS/MS can accurately quantify SA in a high throughtput fashion. The SA linkages can be distinguished by flow cytometry and confocal microscopy with specific lectin labelings. The SA levels and linkages provide markers of cells at different status.

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