Mass Spectrometry Quantitation

National Institute Of Environmental Health Sciences

Investigators

Linked publications & trials

Abstract

We have analyzed a variety of molecules obtained from various sources to get the quantitative information. Additionally, we are developing methods to improve the quantitative information that can be gained. 1. Method Development. We have put effort into the development of lipidomics analyses. This project involves the identification as well as the quantitation of compounds; thus, this project is included in both the identification project and the quantitation project. Currently, we have the framework for a turn-key analysis from sample to data output. However, the method will continue development and refinement to improve upon this initial state, with an eye toward expanding the information it provides and improving where technical advances allow. 2. Eicosanoid Studies. Eicosanoids and related fatty acid metabolites serve as signaling molecules and are intricately involved in inflammation and cardiovascular health. The level of eicosanoids and eicosanoid metabolites are thought to be involved in many diseases. We are involved in a variety of projects measuring these compounds using mass spectrometry. We use liquid chromatography tandem mass spectrometry to analyze a panel of 71 of these molecules which has allowed us to collaborate with several intramural and extramural researchers. We are also developing an untargeted approach to analyze these molecules on another instrument. 3. Bile acid studies. In collaboration with the Zeldin laboratory, we have profiled bile acids in Cyp2c transgenic mice. For this project, we developed LC-MS/MS and SFC-MS/MS methods for profiling of unconjugated and taurine conjugated bile acids. In addition, we developed a derivatization protocol for unconjugated bile acids allowing for superior separation and lower limits of detection. We determined there was platform-dependent variability in the analyses (i.e. greater variance in measurements was seen on a triple quadrupole platform than on a Q-Trap platform). 4. NAD Studies. NAD is a cofactor for hundreds of metabolic reactions in all cell types, and plays an essential role in metabolism, DNA repair, and aging. How NAD metabolism is impacted by the environment remains unclear. Per a request from the laboratory of Dr. Xiaoling Li, the MSRSG has recently developed an LC-MS-based panel for the relative quantitation of metabolites in the NAD pathway. The method was developed to include 8 compounds in the pathway and has been optimized for the analyses of samples arising from cell culture, media, serum, feces, and multiple tissues. We were able to use this approach in the discovery of cooperation between bacteria and mammalian cells wherein bacteria contribute to host NAD biosynthesis. Mechanistically, a microbial nicotinamidase (PncA) can convert nicotinamide to nicotinic acid, a precursor in the alternative deamidated NAD salvage pathway. Using stable isotope tracing and microbiota-depleted mice, we demonstrate that this bacteria-mediated deamidation contributes substantially to the NAD-boosting effect of oral nicotinamide and nicotinamide riboside supplementation in several tissues. Collectively, our findings reveal an important role of bacteria-enabled deamidated pathway in host NAD metabolism. Recently, in collaboration with the Li laboratory, the MSRSG has extended these studies to gene knockout mice as well additional compounds used in isotope tracing studies. 5. Comparative Proteomics Studies. The MSRSG has undertaken several collaborative studies that involve both identification and relative quantification of proteins in samples. Most of these studies have relied on label-free quantification (LFQ) but one noted study employed tandem mass tags (TMT) In collaboration with the Doetsch lab, we have compared the proteins expressed in HBEC lung epithelial cells and MCF10A breast epithelial cells before and after overexpression of Endonuclease III-like protein 1 (NTHL1), a bifunctional DNA glycosylase. Similarly, in collaboration with the Archer laboratory, we have identified proteins from donor-matched fibroblasts and induced pluripotent stem cells as a part of a larger multi-omics project evaluating how genetic ancestry influences reprograming efficiency. Additionally, a similar study evaluating the proteome changes that occur in platelets upon adrenalectomy and dexamethasone treatments is ongoing with the Cidlowski laboratory. Finally, again in collaboration with the Archer laboratory, the MSRSG used TMT-based quantification to evaluate the proteomic changes associated with proteosome inhibition with a particular focus on chromatin remodeling proteins. 6. UDP-Hexose In collaboration with the laboratory of Dr. Don Cook, the MSRSG developed a method for the relative quantitation of UDP-hexose from bronchoalveolar lavage fluid. This method was used to measure UDP-hexose under a variety of challenge conditions. We studied the role of UDP-glucose (UDP-G), a nucleotide sugar that we found to be selectively released into the airways of allergen-sensitized mice upon their subsequent challenge with that same allergen. A manuscript has been submitted to Journal of Clinical Investigation and is in press. Additionally, the MSRSG has performed UDP-hexose quantitation from lavage fluid from mice that have been challenged with polyIC as a surrogate/mimetic of viral and/or SARS-CoV-2 infection. 7. TMT studies. We have used this strategy to investigate possible correlation between plasma proteins and airway dysfunction in established mouse models of LPS-enhanced asthma. These analyses showed that a few proteins of interest were significantly altered in plasma samples originating from LPS-exposed mice. These candidate biomarker proteins are currently further validated by our collaborator from the immunogenic group with orthogonal techniques such as ELISA and Western blotting. 8. Cysteine studies. Methionine restriction, a dietary regimen that protects against metabolic diseases and aging, represses cancer growth and improves cancer therapy. However, the response of different cancer cells to this nutritional manipulation is highly variable, and the molecular determinants of this heterogeneity remain poorly understood. In collaboration with both the Wade lab and the Cui lab, we have measured the effect of methionine restriction in mice.