Non-targeted tracer fate detection

in Methods in Enzymology (2015), 561

Stable isotopes have been used to trace atoms through metabolism and quantify metabolic fluxes for several decades. Only recently non-targeted stable isotope labeling approaches have emerged as a powerful ... [more ▼]

Stable isotopes have been used to trace atoms through metabolism and quantify metabolic fluxes for several decades. Only recently non-targeted stable isotope labeling approaches have emerged as a powerful tool to gain biological insights into metabolism. However, the manual detection of isotopic enrichment for a non-targeted analysis is tedious and time consuming. To overcome this limitation, the non-targeted tracer fate detection (NTFD) algorithm for the automated metabolome-wide detection of isotopic enrichment has been developed. NTFD detects and quantifies isotopic enrichment in the form of mass isotopomer distributions (MIDs) in an automated manner, providing the means to trace functional groups, determine MIDs for metabolic flux analysis, or detect tracer-derived molecules in general. Here, we describe the algorithmic background of NTFD, discuss practical considerations for the freely available NTFD software package, and present potential applications of non-targeted stable isotope labeling analysis. [less ▲]

Sequential isolation of metabolites, RNA, DNA, and proteins from the same unique sample

in Methods in Enzymology (2013), 531

In microbial ecology, high-resolution molecular approaches are essential for characterizing the vast organismal and functional diversity and understanding the interaction of microbial communities with ... [more ▼]

In microbial ecology, high-resolution molecular approaches are essential for characterizing the vast organismal and functional diversity and understanding the interaction of microbial communities with biotic and abiotic environmental factors. Integrated omics, comprising genomics, transcriptomics, proteomics, and metabolomics allows conclusive links to be drawn between genetic potential and function. However, this requires truly systematic measurements. In this chapter, we first assess the levels of heterogeneity within mixed microbial communities, thereby demonstrating the need for analyzing biomolecular fractions obtained from a single and undivided sample to facilitate multi-omic analysis and meaningful data integration. Further, we describe a methodological workflow for the reproducible isolation of concomitant metabolites, RNA (optionally split into large and small RNA fractions), DNA, and proteins. Depending on the nature of the sample, the methodology comprises different (pre)processing and preservation steps. If possible, extracellular polar and nonpolar metabolites may first be extracted from cell supernatants using organic solvents. Cells are homogenized by cryomilling before small molecules are extracted with organic solvents. After cell lysis, nucleic acids and protein fractions are sequentially isolated using chromatographic spin columns. To prove the broad applicability of the methodology, we applied it to microbial consortia of biotechnological (biological wastewater treatment biomass), environmental (freshwater planktonic communities), and biomedical (human fecal sample) research interest. The methodological framework should be applicable to other microbial communities as well as other biological samples with a minimum of tailoring and represents an important first step in standardization for the emerging field of Molecular Eco-Systems Biology. [less ▲]

GTPases and the control of neuronal polarity.

in Methods in Enzymology (2006), 406

Neurons are probably the most highly polarized cell type and typically develop a single axon and several dendrites. The establishment of a polarized morphology and the functional specialization of axonal ... [more ▼]

Neurons are probably the most highly polarized cell type and typically develop a single axon and several dendrites. The establishment of a polarized morphology and the functional specialization of axonal and dendritic compartments are essential steps in the differentiation of neurons. Primary cultures of dissociated hippocampal neurons are a widely used system to study the development of neuronal differentiation. In this article, we will describe gain-of-function and loss-of-function approaches that allow us to analyze the role of GTPases in neuronal differentiation. [less ▲]