MassIVE MSV000097408

Partial Public PXD062133

Guard Cell Mitochondria Prefer Alternative Substrates and Energy-Conserving Electron Transfer

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Description

Proteomics of plant mitochondria usually is based on a bulk mixture of organelles originating from different leaf cell types. Here, a novel isolation technique was applied, fusing an affinity tag under control of cell type specific promotors to a mitochondrial outer envelope protein. The construct allowed isolation of mitochondria from the mesophyll, guard cells, and total leafs, serving as a control. Isolated organelles were subjected to a shotgun MS workflow employing single pot, solid phase enhanced sample preparation (SP3, Hughes et al. 2019) protocol. Tryptic peptides were separated using a 60 min gradient on a Aurora 25cm column, connected to a nanoElute2 LC that was coupled to a timsTOF Pro MS. Spectra were acquired in DDA-PASEF and used to compute intensity based abundance quantification (iBAQ, Schwanhausser et al. 2011) values to assess organelle purity. Total leaf protein extracts were analyzed to estimate enrichment factors for mitochondrial proteins. Label free quantification values were used to compare the mitochondrial proteomes originating from different leaf tissues. Additionally, differences of mitochondrial protein complexes were investigated by complexome profiling. For this, mitochondria from different tissues were fractionated by blue native polyacrylamide gel electrophoresis, resulting in 39 fractions for each tissue that were subjected to tryptiv in-gel digestion and nanoElute2-timsTOF-MS to generate abundance profiles from iBAQ values. Last, abundance of complex I was estimated to calculate its NADH oxidase activity between the different tissues. For this, an Evosep was operated using the Whisper Zoom 40 SPD, coupled to a timsTOF Pro operating in DDA-PASEF. Abundance was estimated using iBAQ values of identified complex I subunits and their contribution to the summed up iBAQ intensity within each sample. [doi:10.25345/C5QB9VJ2C] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: guard cell, mitochondria, complexome profiling, photorespiration, amino acid metabolism, OXPHOS ; DatasetType:Proteomics

Contact

Principal Investigators:
(in alphabetical order) 		Holger Eubel, Leibniz University Hannover, Germany
Submitting User: NoahDitz
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