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 obtain quantitative information and are developing methods to improve this process. Method Development: We have focused on developing lipidomics analyses, which involve both the identification and quantitation of compounds. This project, therefore, falls under both identification/characterization and quantitation categories. We have established a framework for turn-key analysis from sample processing to data output. In collaboration with the Cidlowski, Guardia, and Kurtz laboratories, we have been analyzing lipid profiles from animal tissues, cells, and rodent feeds. We can currently distinguish and annotate many hundreds of lipids, although the isomeric nature of lipids limits the exactness of the assay. We continue to work with Dr. Alan Jarmusch to implement statistical methods for handling the large amounts of data produced by this approach. These novel, custom-built tools allow us to provide relative, semi-quantitative measurements for several hundred individual features. Eicosanoid Studies: Eicosanoids and related fatty acid metabolites act as signaling molecules involved in inflammation and cardiovascular health. Their levels are implicated in many diseases. We measure these compounds using liquid chromatography tandem mass spectrometry (LC-MS/MS), analyzing a panel of about 75 unique molecules. This has facilitated collaborations with several intramural and extramural researchers. Bile Acid Studies: In collaboration with the Zeldin, Shaw, and Garantziotis laboratories, we have profiled bile acids in mice and humans. 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. We are continuing our collaboration with the Li laboratory to extend these studies to gene knockout mice and additional compounds and flux analyses. Comparative Proteomics Studies: The MSRSG has undertaken several collaborative studies involving both identification and relative quantification of proteins in samples. While some studies use tandem mass tags (TMT), most rely on label-free quantification (LFQ). For example, in collaboration with the Cidlowski laboratory, we evaluated proteome changes in murine platelets following adrenalectomy and dexamethasone treatments. Similarly, with Drs. Wendy Jefferson and Carmen Williams, we quantified proteins from uterine tissue of WT and PADI KO mice as a function of the estrus cycle and DES treatment. Additionally, with Drs. Y Liu and P. Wade, we evaluated protein expression profiles in the livers of mice fed a normal diet versus those undergoing methionine deprivation. TMT Studies: This method uses stable isotope labels incorporated within peptides, introducing a mass difference across experimental conditions, thus increasing sample throughput and enabling relative quantitation of proteins. Recently, we quantified IgA-containing protein complexes in the circulation of IgAN patients and controls using TMT-11 reagents. Minimal sample preparation was used, requiring only one-eighth of the starting material recommended by the manufacturer. In total, 116 samples, including replicates and triplicates, were analyzed, identifying 195 proteins and robustly quantifying 151, including one significantly upregulated protein in patient samples. Neurosteroid Studies: In collaboration with Dr. Serena Dudek's laboratory, we investigated the impact of prenatal corticosterone exposure on neurosteroid levels in mouse pups using advanced mass spectrometry techniques. Pregnant dams received corticosterone in their drinking water, and tissue samples from pups were collected at various developmental stages. These findings could provide insights into biochemical pathways affected by prenatal stress and their implications for neurodevelopmental outcomes. TCA and Glycolysis Intermediate Measurements: In collaboration with Dr. Traci Hall and others, we measured metabolites in the TCA and glycolytic pathways to understand the role of the essential Drosophila RNA-binding protein Brain Tumor (Brat). We found that Brat depletion affected many mRNAs with enriched 3′UTR motifs matching Brat binding sites with many of the corresponding proteins involved in glycolysis and the vacuolar ATPase complex. These observations, along with our metabolite measurements, linked Brat to metabolism and intracellular pH regulation. Additionally, the MSRSG has developed specialized sample preparation and mass spectrometric techniques to successfully detect and quantitate a variety of compounds in multiple research projects (e.g., NE, NAR, PGPC, etc.).