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. 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 75 of these molecules which has allowed us to collaborate with several intramural and extramural researchers. 3. Bile acid studies. In collaboration with the Zeldin laboratory, we have profiled bile acids in mice. For this project, we examined cyp450 enzymes for bile acid metabolism. Positive control cyp2C70 formed alpha-MCA from CDCA but 2c44 and 2c29 did not. 2c44 microsome activity was confirmed which showed arachidonic acid being converted to oxylipin species. 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. 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 example, in collaboration with the Cidlowski laboratory, we have performed a study evaluating the proteome changes that occur in murine platelets upon adrenalectomy and dexamethasone treatments. Similarly, in collaboration with Carol Trempus and Dr. S. Garantziotis, we have performed a relative quantification of proteins from the PDGFRalpha positive fibroblasts harvested from the lungs of PBS and bleomycin treated mice. Additionally, in collaboration with Drs. Y Liu and P. Wade, we have performed proteomic studies to evaluate the protein expression profiles in the livers of mice that have been fed a normal diet versus mice that have undergone methionine deprivation. Finally, in collaboration with Drs. E. Haque and S. London, we have evaluated protein expression profiles in the lungs of mice exposed to cigarette smoke versus mice that have not undergone such treatment. 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 used the approach to quantify IgA-containing protein complexes in circulation of IgAN patients and controls. IgA protein complexes were enzymatically digested, and the resulting peptides were labelled with TMT-11 reagent. Differences in IgA-protein complex abundance were quantified by combining TMT-labeled peptides from patients and controls and analyzing the peptide simultaneously by LC-MS/MS. A minimalistic sample preparation strategy was chosen to streamline TMT sample preparation. The protocol consumes only one-eighth of the starting material (12.5g plasma proteins) recommended by the company that manufactures TMT, and thus used the proportional reduced amount of the labeling reagent. Each TMT-11 set included 1 pooled control sample for across TMT-plex normalizations, allowing up to 10 biological samples to be analyzed in a single mass spectrometry experiment (Figure 1). In total, 116 samples (12 injections), including replicates and triplicates, were analyzed. 195 proteins were identified, and 151 proteins were robustly quantified, in the tryptic digest of the 2 types of samples, including one significantly up regulated protein in the patient samples that, according to a recent publication, showed strong positive correlation with mesangial cell injury parameters in IgAN patients. 7. Methionine deprivation 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 adenosine. We have performed analyses looking at N-6-methyldeoxyadenosine incorporation into DNA for the Wade lab. A new chromatographic regime was employed for this assay, using a mixed bed C18-Penta-flouro phenyl (PFP) column. New chromatographic methods will explore the feasibility of assaying phosphorylated nucleoside and deoxynucleoside species in an effort to expand and increase the sensitivity of the assay. 9. Buprenorphine Study. Buprenorphine is an opiate which is used to control pain in experimental animals by our CMB. Plasma concentrations of buprenorphine drop below therapeutic levels between 2 and 4 hours after subcutaneous injection. We initially reported the results from extended-release formulations out to 24 hours. We recently acquired data from samples out to 48 hours. In addition, we are also acquiring data to compare bup SR-LAB to the new extended release product Zorbium. 10. Acrylamide studies. We recently obtained acrylamide levels in feed resulting from dry heat sterilization to compare to our previously obtained unsterilized, irradiated, and autoclaved feed. We determined that sterilizing dry heat conditions created an order of magnitude increase in acrylamide content compared to autoclave sterilization. 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. ppGpp, acylCoAs, etc).