Formation and Fragmentation of Unsaturated Fatty Acid M-2H+Na (-) Ions: Stabilized Carbanions for Charge-Directed Fragmentation

Fatty acids are long-chain carboxylic acids that readily produce \[M - H](-) ions upon negative ion electrospray ionization (ESI) and cationic complexes with alkali, alkaline earth, and transition metals in positive ion ESI. In contrast, only one anionic monomeric fatty acid-metal ion complex has been reported in the literature, namely \[M - 2H + (FeCl)-Cl-II](-). In this manuscript, we present two methods to form anionic unsaturated fatty acid-sodium ion complexes (i.e., \[M - 2H + Na](-)). We find that these ions may be generated efficiently by two distinct methods: (1) negative ion ESI of a methanolic solution containing the fatty acid and sodium fluoride forming an \[M - H + NaF](-) ion. Subsequent collision-induced dissociation (CID) results in the desired \[M - 2H + Na](-) ion via the neutral loss of HF. (2) Direct formation of the \[M - 2H + Na](-) ion by negative ion ESI of a methanolic solution containing the fatty acid and sodium hydroxide or bicarbonate. In addition to deprotonation of the carboxylic acid moiety, formation of \[M - 2H + Na](-) ions requires the removal of a proton from the fatty acid acyl chain. We propose that this deprotonation occurs at the bis-allylic position(s) of polyunsaturated fatty acids resulting in the formation of a resonance-stabilized carbanion. This proposal is supported by ab initio calculations, which reveal that removal of a proton from the bis-allylic position, followed by neutral loss of HX (where X = F- and -OH), is the lowest energy dissociation pathway

Established and proposed roles of xanthine oxidoreductase in oxidative and reductive pathways in plants

Xanthine oxidoreductase (XOR) is among the most-intensively studied enzymes known to participate in the consumption of oxygen in cells. However, it attracted the attention of researchers due its participation in free radical production in vivo, mainly through the production of superoxide radicals. In plants, XOR is a key enzyme in purine degradation where it catalyzes the oxidation of hypoxanthine to xanthine and of xanthine to uric acid. Both reactions are accompanied by electron transfer to either NAD+ with simultaneous formation of NADH or to molecular oxygen, which results in formation of superoxides. Characterization of plant XOR mutants and isolated XOR proteins from various plant species provided evidence that the enzyme plays significant roles in plant growth, leaf senescence, fruit size, synthesis of nitrogen storage compounds, and plant-pathogen interactions. Moreover, the ability of XOR to carry out redox reactions as NADH oxidase and to produce reactive oxygen species and nitric oxide, together with a possible complementary role in abscisic acid synthesis have raised further attention on the importance of this enzyme. Based on these established and proposed functions, XOR is discussed as regulator of different processes of interest in plant biology and agriculture.The authors acknowledge the support of the research grants AGL2010-16167 to J.F.M. from the Spanish Ministry of Science and Innovation and Bi 1075/5-1 to F.B. by the Deutsche Forschungsgemeinschaft. R.E. received a JAE-Doctor grant from the Spanish Research Council (CSIC).

Time to face the fats: What can mass spectrometry reveal about the structure of lipids and their interactions with proteins?

Free to read at publisher Since the 1950s, X-ray crystallography has been the mainstay of structural biology, providing detailed atomic-level structures that continue to revolutionize our understanding of protein function. From recent advances in this discipline, a picture has emerged of intimate and specific interactions between lipids and proteins that has driven renewed interest in the structure of lipids themselves and raised intriguing questions as to the specificity and stoichiometry in lipid-protein complexes. Herein we demonstrate some of the limitations of crystallography in resolving critical structural features of ligated lipids and thus determining how these motifs impact protein binding. As a consequence, mass spectrometry must play an important and complementary role in unraveling the complexities of lipid-protein interactions. We evaluate recent advances and highlight ongoing challenges towards the twin goals of (1) complete structure elucidation of low, abundant, and structurally diverse lipids by mass spectrometry alone, and (2) assignment of stoichiometry and specificity of lipid interactions within protein complexes