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J Proteome Res. 2015 Sep 4;14(9):3900-11. doi: 10.1021/acs.jproteome.5b00329. Epub 2015 Aug 10.

Linear Discriminant Analysis Identifies Mitochondrially Localized Proteins in Neurospora crassa.

Author information

1
Cellular Proteomics Research Group, §Central Facility for Microscopy, Helmholtz Centre for Infection Research , 38124 Braunschweig, Germany.
2
Department of Computer Science, Ostfalia University of Applied Sciences , 38302 Wolfenbüttel, Germany.

Abstract

Besides their role as powerhouses, mitochondria play a pivotal role in the spatial organization of numerous enzymatic functions. They are connected to the ER, and many pathways are organized through the mitochondrial membranes. Thus, the precise definition of mitochondrial proteomes remains a challenging task. Here, we have established a proteomic strategy to accurately determine the mitochondrial localization of proteins from the fungal model organism Neurospora crassa. This strategy relies on both highly pure mitochondria as well as the quantitative monitoring of mitochondrial components along their consecutive enrichment. Pure intact mitochondria were obtained by a multistep approach combining differential and density Percoll (ultra) centrifugations. When compared with three other intermediate enrichment stages, peptide sequencing and quantitative profiling of pure mitochondrial fractions revealed prototypic regulatory profiles of per se mitochondrial components. These regulatory profiles constitute a distinct cluster defining the mitochondrial compartment and support linear discriminant analyses, which rationalized the annotation process. In total, this approach experimentally validated the mitochondrial localization of 512 proteins including 57 proteins that had not been reported for N. crassa before.

KEYWORDS:

Mitochondrial proteomics; Neurospora crassa; d-arabinitol dehydrogenase; heme biosynthesis; linear discriminant analysis (LDA)

PMID:
26215788
DOI:
10.1021/acs.jproteome.5b00329
[Indexed for MEDLINE]

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