Mass Spectrometry Characterization

National Institute Of Environmental Health Sciences

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Abstract

Mass spectrometry has been used to determine the extent of modification and the specific sites of modification on biomolecules. MS-based approaches have many advantages, including generally rapid analyses without radiolabeling. The MS analysis of protein-DNA crosslinks has been investigated using mass spectrometry. Products and digests have been analyzed by both positive and negative ion MALDI mass spectrometry and LC in combination with electrospray mass spectrometry. Additionally, products following crosslinking have been purified by SDS-PAGE and investigated by mass spectrometry. Free radicals are implicated in oxidative stress and are associated with a wide range of diseases and disorders. In this work, we have investigated the sites of radicals trapped by DMPO on deoxyribonucleosides and proteins using LC/MS/MS. A recent undertaking is the qualitative analyses of proteins present in dust. This project is in collaboration with Dr. Geoff Mueller and Dr. Don Cook and the PMCF. Dr. Cook has demonstrated that there is considerable variation in the adjuvant activity in various house dust extracts, as measured by their ability to promote allergic sensitization to ovalbumin, which on its own, is inert. Interestingly, there is not a direct correlation in the ability of the extracts to promote Th2 and Th17 responses, as inferred from eosinophil and neutrophil responses, respectively. We are interested in determining if any protein in the various extracts is associated with eosinophilic response in particular. In these efforts, qualitative analyses have been performed on 8 dust samples thus far using GeLC-MS/MS approaches. Not surprisingly, proteins from humans dominate the samples but known allergens from dust mites and domestic animals have also been observed. Pilot studies have been initiated on the quantitative analyses, but results are still preliminary. Our aim is to quantify the proteins found in these house dust extracts and attempt to correlate individual protein abundances with eosinophil response.