Cell Biology

Lysine acetylation is a conserved post-translational modification and a key regulatory mechanism for various cellular processes, including metabolic control, epigenetic regulation, and cellular signaling transduction. Recent advances in mass spectrometry (MS) enable the extensive identification of acetylated lysine residues of histone and non-histone proteins. However, protein enrichment before MS analysis may be necessary to improve the detection of low-abundant proteins or proteins that exhibit low acetylation levels. Fatty acid synthase (FASN), an essential enzyme catalyzing the de novo synthesis of fatty acids, has been found to be acetylated in various species, from fruit flies to humans. Here, we describe a step-by-step process of antibody-based protein enrichment and sample preparation for acetylation identification of endogenous FASN protein by MS-based proteomics analysis. Meanwhile, we provide a protocol for nicotinamide adenine dinucleotide phosphate (NADPH) absorbance assay for FASN activity measurement, which is one of the primary functional readouts of de novo lipogenesis.

Key features

• A comprehensive protocol for protein immunoprecipitation and sample preparation for acetylation site identification by mass spectrometry.

• Step-by-step procedures for measurement of FASN activity of fruit fly larvae using an absorbance assay.

Graphical overview

Once thought to be a mere consequence of the state of a cell, intermediary metabolism is now recognized as a key regulator of mammalian cell fate and function. In addition, cell metabolism is often disturbed in malignancies such as cancer, and targeting metabolic pathways can provide new therapeutic options. Cell metabolism is mostly studied in cell cultures in vitro, using techniques such as metabolomics, stable isotope tracing, and biochemical assays. Increasing evidence however shows that the metabolic profile of cells is highly dependent on the microenvironment, and metabolic vulnerabilities identified in vitro do not always translate to in vivo settings. Here, we provide a detailed protocol on how to perform in vivo stable isotope tracing in leukemia cells in mice, focusing on glutamine metabolism in acute myeloid leukemia (AML) cells. This method allows studying the metabolic profile of leukemia cells in their native bone marrow niche.

Two enantiomers of 2-hydroxyglutarate (2HG), L (L2HG) and D (D2HG), are metabolites of unknown function in mammalian cells that were initially associated with separate and rare inborn errors of metabolism resulting in increased urinary excretion of 2HG linked to neurological deficits in children (Chalmers et al., 1980; Duran et al., 1980; Kranendijk et al., 2012). More recently, investigators have shown that D2HG is produced by mutant isocitrate dehydrogenase enzymes associated with a variety of human malignancies, such as acute myeloid leukemia, glioblastoma multiforme, and cholangiocarcinoma (Cairns and Mak, 2013; Dang et al., 2009; Ward et al., 2010). By contrast, we and others have shown that L2HG accumulates in response to cellular reductive stressors like hypoxia, activation of hypoxia inducible factors, and mitochondrial electron transport chain defects (Oldham et al., 2015; Reinecke et al., 2011; Intlekofer et al., 2015; Mullen et al., 2015). Each enantiomer is produced and metabolized in independent biochemical pathways in reactions catalyzed by separate enzymes and utilizing different cofactors with presumably different consequences for cellular metabolism (Kranendijk et al., 2012). Therefore, as research into the roles of D2HG and L2HG in human metabolism continues, it becomes increasingly important for investigators to consider each enantiomer independently (Struys, 2013). Several methods for quantification of biochemically relevant enantiomers in general have been developed and typically include enzymatic assays using enzymes specific for one enantiomeric species or the other, the use of chiral chromatography medium to facilitate chromatographic separation of enantiomers prior to spectroscopy, or the use of chiral derivatization reagents to convert a mixture of enantiomers to diastereomers with differing physical and chemical properties facilitating their chromatographic separation. In this protocol, we report the adaptation of a previously published derivatization method using diacetyl-L-tartaric anhydride (DATAN) for the quantification of 2HG enantiomers (Figure 1) (Oldham et al., 2015; Struys et al., 2004).


Figure 1. Reaction scheme for the derivatization protocol

Evidence of the involvement of tryptophan and its metabolite, kynurenine, in various biological processes including cancer is constantly expanding. Analysis of cell extracts and culture media can allow for quick snapshots of the metabolic fluctuations occurring in vitro. Here, we describe a method for metabolite extraction from mammalian cells and analysis of extracted metabolites and cell culture media by HPLC with detection using an ultra-sensitive diode array detector.

Nicotinamide adenine dinucleotide (NAD⁺) is a coenzyme for many NAD⁺-consuming proteins with diverse biological functions. Oscillations in NAD⁺ levels may influence several cellular signaling pathways. NAD⁺ synthesis via Preiss-Handler route (salvage reactions) has been extensively reported. However, the contribution of L-tryptophan/kynurenine catabolism in de novo NAD⁺ synthesis is poorly understood. Using L-[14C]-tryptophan tracing in four liver cancer cell lines and siRNA-mediated silencing of arylformamidase (AFMID), a key enzyme involved in L-tryptophan degradation, we demonstrate the contribution of L-tryptophan catabolism in de novo synthesis of NAD⁺ pools. NAD⁺ modulation is therefore important in maintaining cellular homeostasis and appropriate cellular functions according to nutrients availability.