Chronic caloric restriction partially protects against age-related alteration in serum metabolome.

Calorie restriction (CR) remains the most robust metabolic intervention to extend lifespan and improve healthspan in several species. Using global and targeted mass spectrometry-based metabolomics approaches, here we show that chronic CR prevents age-related changes in specific metabolic signatures. Global metabolomic analysis using ultra-performance liquid chromatography-tandem mass spectrometry detected more than 7,000 metabolites in sera from ad-libitum-fed young, aged, and aged C57BL/6 mice maintained on 40 % CR. Multivariate statistical analysis of mass spectrometry data revealed a clear separation among the young, aged, and aged-CR mice demonstrating the potential of this approach for producing reliable metabolic profiles that discriminate based on age and diet. We have identified 168 discriminating features with high statistical significance (p ≤ 0.001) and validated and quantified three of these metabolites using targeted metabolite analysis. Calorie restriction prevented the age-related alteration in specific metabolites, namely lysophosphatidylcholines (16:1 and 18:4), sphingomyelin (d18:1/12:0), tetracosahexaenoic acid, and 7α-dihydroxy-4-cholesten-3-one, in the serum. Pathway analysis revealed that CR impacted the age-related changes in metabolic byproducts of lipid metabolism, fatty acid metabolism, and bile acid biosynthesis. Our data suggest that metabolomics approach has the potential to elucidate the metabolic mechanism of CR's potential anti-aging effects in larger-scale investigations.

A novel LC-MS approach for the detection of metabolites in DMPK studies.

BACKGROUND: The profiling and quantification of drug metabolites in discovery and development bioanalysis studies is playing an increasingly important role in early candidate selection. Using a conventional tandem quadrupole mass spectrometer this activity normally requires several analytical runs to acquire the necessary analytical data. RESULTS: In this article we present the use of a new tandem quadrupole mass spectrometer equipped with a novel collision cell design, which allows the rapid switching between multiple reaction monitoring and full-scan MS mode. This approach allowed for the collection of multiple reaction monitoring data and full-scan data with no loss in sensitivity, with analysis times in the 1-2 min range. CONCLUSION: A modified approach of using the multiple reaction monitoring data to trigger the acquisition of full scan MS/MS data is described, where the data is collected on the trailing edge of the LC-MS peak, thus improving data quality and throughput.

Semi-automated data processing of hydrogen exchange mass spectra using HX-Express.

A Microsoft Excel utility, HX-Express, that significantly accelerates the analysis of hydrogen exchange mass spectrometry data is described. HX-Express generates deuterium uptake and peak width plots from peaks in mass spectral data. Data analysis is intentionally semi-automated, requiring that the user find the peaks to be analyzed. The peaks are entered in the form of x, y lists of m/z versus intensity or can be directly imported from Waters MassLynx software. Analysis of data with HX-Express provides the same results as manual data processing but is substantially faster. In addition to speed, HX-Express provides and preserves visual and numeric displays of the analysis process for quality control.

Evaluation of multidimensional (ion-exchange/reversed-phase) protein separations using linear and step gradients in the first dimension.

The performance characteristics of multidimensional liquid chromatographic protein separations were evaluated using on-line electrospray mass detection, and a novel workflow for automated LC/MS data processing. Two-dimensional ion exchange/reversed-phase LC separations of Escherichia coli cytosol were conducted using either a continuous linear or discontinuous step gradient in the first dimension. Chromatographic profiles of the top 100 most abundant components were characterized to assess overall separation reproducibility within each mode, and to characterize differences in component distribution between the two modes of operation. Analysis of the resulting data indicates that multidimensional separations of complex protein mixtures can be done reproducibly. Furthermore, under the conditions employed within this study, a linear first dimension gradient was more effective at fractionating the protein mixture, distributing fewer major components to multiple second dimension cycles than an equivalent step gradient. The application of on line mass spectrometry, and automated processing of the resulting data, proved valuable for producing component level analysis of multidimensional protein separations.

Quantitative proteomic analysis by accurate mass retention time pairs.

Current methodologies for protein quantitation include 2-dimensional gel electrophoresis techniques, metabolic labeling, and stable isotope labeling methods to name only a few. The current literature illustrates both pros and cons for each of the previously mentioned methodologies. Keeping with the teachings of William of Ockham, "with all things being equal the simplest solution tends to be correct", a simple LC/MS based methodology is presented that allows relative changes in abundance of proteins in highly complex mixtures to be determined. Utilizing a reproducible chromatographic separations system along with the high mass resolution and mass accuracy of an orthogonal time-of-flight mass spectrometer, the quantitative comparison of tens of thousands of ions emanating from identically prepared control and experimental samples can be made. Using this configuration, we can determine the change in relative abundance of a small number of ions between the two conditions solely by accurate mass and retention time. Employing standard operating procedures for both sample preparation and ESI-mass spectrometry, one typically obtains under 5 ppm mass precision and quantitative variations between 10 and 15%. The principal focus of this paper will demonstrate the quantitative aspects of the methodology and continue with a discussion of the associated, complementary qualitative capabilities.