Date of Graduation

5-2015

Document Type

Thesis

Degree Name

Master of Science in Chemistry (MS)

Degree Level

Graduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Stenken, Julie A.

Committee Member

Lay, Jackson O. Jr.

Second Committee Member

Paul, David W.

Third Committee Member

Thallapuranam, Suresh

Keywords

LC-MS; MALDI-MS; Macrophage activation; Mass spectrometry; Proteins

Abstract

Macrophages are versatile and highly adaptive cells that are involved in a wide range of physiological processes including host defense, homeostasis or regeneration, as well as pathogenesis. They react to their microenvironment, assuming various roles based on chemical and/or physical cues, and can reversibly shift between these so-called activation states. Concurrently, the technique of immunohistochemistry is used to gain spatial information on activated macrophages on tissue sections. The aim of this work was to find mass spectral biomarkers that allow the differentiation of activation states, and establish conditions that can be used in imaging mass spectrometry (IMS) experiments to investigate the spatial distribution of differently-activated macrophages within tissue sections. The immortalized macrophage line NR8383 (alveolar, rat) was used, and in vitro activated with the endotoxin lipopolysaccharide (LPS), or with the cytokine interleukin-4 (IL-4). In IMS, tissue sections are commonly prepared to be compatible with matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS) experiments. The tested combinations of MALDI preparation techniques and instrument parameters however remained unsuccessful at distinguishing activation states. Through lowering the complexity of the sample with a 30 minute high-performance liquid chromatography (HPLC) separation, a reproducible biomarker for LPS-activation could be found in electrospray ionization (ESI) MS experiments. The isolated biomarker was subjected to a tryptic digestion, and the resulting tryptic fragments analyzed by MALDI MS. A MASCOT database search suggested the macrophage-capping protein (CAPG) as source of the peptide, which could be validated through peptide sequencing through post-source decay experiments conducted on the tryptic fragments

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