Cristea - CobB PRM

Cristea - CobB PRM
The conservation of sirtuin lipoamidase activity in bacteria as a regulator of metabolic enzyme complexes

  • Organism: E. coli
  • Instrument: Q-Exactive, Orbitrap Velos
  • SpikeIn: No
Abstract
Sirtuins are an essential family of nicotinamide adenine dinucleotide (NAD)-dependent enzymes, conserved from bacteria to humans. Here we study the bacterial sirtuin CobB in E. coli. To demonstrate its highly conserved enzymatic activities and substrates, we used targeted mass spectrometry to quantify CobB levels, lipoylation levels, protein interactions, and the significance of its function in various nutrient conditions.
Experiment Description
The experiments were completed using MC4100 E. coli (wild type, ΔcobB, and CobB overexpression via an IPTG-inducible promoter), and analyzed in triplicate. Total area values were exported from Skyline and normalized by the average MS1 intensity per run calculated using RawMeat. (1) CobB protein levels were measured by SRM-MS using an Orbitrap Velos. (2) Site-specific lipoylation levels of proteins ODP2 and GCSH were measured in wild type and ΔcobB cells using an Orbitrap Velos. (3) Protein interactions with His-CobB or IgG were quantified by PRM-MS using a Q-Exactive. (4) Protein levels were measured using PRM-MS with a Q-Exactive after growing cells in media with different carbon sources.
Sample Description
(1) CobB levels: Wild type and ΔcobB cells were analyzed in the file “CobB_ProteinLevels_WT_Delta_20150811.” IPTG-induced and non-induced cells were analyzed in the file “CobB_ProteinLevels_IPTG_20150805” (2) Lipoyl modification levels: “Lipoylation_SRM_MC4100_WTvDeltaCobB_20151015” (3) CobB Protein Interactions: His-CobB expressing cells were analyzed in the file “His_IP_PRM_20170414” (4) CobB and substrate protein levels in varying carbon sources: Wild type and ΔcobB cells were analyzed in the file “Carbon_Sources_PRM_MC4100_WTvDeltaCobB_20170328”
Created on 9/27/17, 2:01 PM
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