Mass Spectrometry Quantitation

National Institute Of Environmental Health Sciences

Investigators

Linked publications, trials & patents

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/characterization project and the quantitation project. Currently, we have the framework for a turn-key analysis from sample to data output. In collaboration with the Harvey lab at NIDA, this was utilized for the analysis of membrane lipid composition following treatment of neuroblastoma SH-SY5Y cells with palmitate. We were able to show a change in the PC:PE ratio upon treatment. 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. We currently are able to distinguish and annotate many hundreds of lipids, however the isomeric nature of lipids limits the exactness of the assay due to this theoretical possibility. We have been working with Alan Jarmusch to implement statistical methods to deal with the large amounts of data that mass spec can provide. These are novel custom-built tools, not relying on software packages, for in-house statistical analysis. This allows us to provide relative, semi-quantitative measurements for several hundred or more individual features. 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. 3. Bile acid studies. For this project, we have derivatized and analyzed a bile acid set of 26 liver samples with 26 matched plasma samples. In addition, the derivatized free bile acids were monitored in the positive ion mode and the tauro-conjugates were monitored in the negative ion mode. Derivatization and LC-MS/MS parameters were previously developed in house. 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 recently developed an LC-MS-based panel for the relative and/or absolute quantitation of metabolites in the NAD pathway. The method was developed to include multiple compounds in the pathway and has been optimized for the analyses of samples arising from a variety of biological matrices. Recently, in the continuing collaboration with the Li laboratory, the MSRSG has extended these studies to gene knockout mice as well additional compounds and flux analyses. 5. Comparative Proteomics Studies. The MSRSG has undertaken several collaborative studies that involve both identification and relative quantification of proteins in samples. While some studies may employ tandem mass tags (TMT), most of these studies have relied on label-free quantification (LFQ). As examples, 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. 6. TMT studies. This well-established method utilizes stable isotope labels which are incorporated within the peptides, introducing an expectable mass difference within two or more experimental conditions. The strategy considerably increases sample throughput and enables relative quantitation of proteins in samples derived from cells, tissues or biological fluids, thus saving time by reducing the number of mass spectrometry runs required for analysis. We have recently examined the analysis of a TMT11plex standard. We are currently exploring the capabilities of a 16plex TMT strategy with our existing instrumentation. 7. 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 the Wade lab, we have measured the effect of methionine restriction on sulfur metabolism in mice. 8. Nucleosides. The nucleoside methods have been available for some time for non-phosphorylated ribonucleosides, but this year we have expanded the assay to include some modified forms of cytidine. We have returned data to the Archer lab. There has been interest in analyzing phosphorylated forms, so we have been working on expanding the assay to include those. However, this will entail completely new methodology, mostly from the ground up. So it will take time to accomplish a basic working assay.