Lipoyl synthase (LIAS) is an iron–sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe–4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, respectively. Most information concerning LIAS and its mechanism has been determined from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron–sulfur (Fe–S) cluster reconstitution mechanisms. Cluster donation by a number of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe–2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liquid chromatography–mass spectrometry (LC–MS) assay. Electron paramagnetic resonance (EPR) studies of native LIAS and substituted derivatives that lacked the ability to bind one or the other of LIAS’s two [4Fe–4S] clusters revealed a likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe–4S] center. These results detail the trafficking of Fe–S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe–S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe–4S] cluster reconstitution is evident.
Lipids are essential macromolecules that play a crucial role in numerous biological events. Lipids are structurally diverse which allows them to fulfill multiple functional roles. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a powerful tool to understand the spatial localization of lipids within biological systems. Herein, we report the use of ammonium fluoride (NH 4 F) as a comatrix additive to enhance lipid detection in biological samples, with a signal increase of up to 200%. Emphasis was placed on anionic lipid enhancement with negative polarity measurements, with some preliminary work on cationic lipids detailed. We observed lipid signal enhancement of [M−H] − ions with the addition of NH 4 F additive attributed to a proton transfer reaction in several different lipid classes. Overall, our study demonstrates that the use of the NH 4 F comatrix additive substantially improves sensitivity for lipid detection in a MALDI system and is capable of being applied to a variety of different applications.
Lipids are essential macromolecules that play a crucial role in numerous biological events. Lipids are structurally diverse which allows them to fulfill multiple functional roles. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a powerful tool to understand the spatial localization of lipids within biological systems. Herein, we report the use of ammonium fluoride (NH4F) as a comatrix additive to enhance lipid detection in biological samples, with signal increase up to 200%. Emphasis was placed on anionic lipid enhancement with negative polarity measurements, with some preliminary work on cationic lipids detailed. We observed lipid signal enhancement of [M-H]- ions with the addition of 500 uM NH4F additive attributed to a proton transfer reaction in several different lipid classes. Overall, our study demon-strates that the use of NH4F comatrix additive substantially improves sensitivity for lipid detection in a MALDI system.
Lipids are essential macromolecules that play a crucial role in numerous biological events. Lipids are structurally diverse which allows them to fulfill multiple functional roles. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a powerful tool to understand the spatial localization of lipids within biological systems. Herein, we report the use of ammonium fluoride (NH4F) as a comatrix additive to enhance lipid detection in biological samples, with signal increase up to 200%. Emphasis was placed on anionic lipid enhancement with negative polarity measurements, with some preliminary work on cationic lipids detailed. We observed lipid signal enhancement of [M-H]- ions with the addition of 500 uM NH4F additive attributed to a proton transfer reaction in several different lipid classes. Overall, our study demonstrates that the use of NH4F comatrix additive substantially improves sensitivity for lipid detection in a MALDI system.
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