Proteomics

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Mass spectrometry approach to identify mitochondrial poly(A) RNA interacting proteins

ABSTRACT: To identify mitochondrial RNA-interacting proteins, we developed three related mass spectrometry based methods. In the first method, called mitochondrial crosslinking (MXL), mitochondria of 4-thiouridine labelled cells were isolated, exposed to UV-light and poly(A) RNA was isolated. In the second method, called whole cell crosslinking (WCXL), 4-thiouridine labelled cells were first exposed to UV-light, subsequently mitochondria were isolated followed by a poly(A) RNA extraction. The third method, called ethidium bromide whole cell crosslinking (WCXL_EtBr), is exactly the same as the WCXL method, but the cells are cultured with ethidium bromide to reduce the amount of mitochondrial RNA. In all methods poly(A) RNA was degraded and proteins in the sample were digested and identified using mass spectrometry.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Selma van Esveld  

LAB HEAD: Johannes N. Spelbrink

PROVIDER: PXD014957 | Pride | 2019-10-15

REPOSITORIES: Pride

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Dataset's files

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MXL1_1_NoXL1.raw Raw
MXL1_1_NoXL2.raw Raw
MXL1_1_NoXL3.raw Raw
MXL1_2_XL1.raw Raw
MXL1_2_XL2.raw Raw
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Publications

A Combined Mass Spectrometry and Data Integration Approach to Predict the Mitochondrial Poly(A) RNA Interacting Proteome.

van Esveld Selma L SL   Cansız-Arda Şirin Ş   Hensen Fenna F   van der Lee Robin R   Huynen Martijn A MA   Spelbrink Johannes N JN  

Frontiers in cell and developmental biology 20191115

In order to synthesize the 13 oxidative phosphorylation proteins encoded by mammalian mtDNA, a large assortment of nuclear encoded proteins is required. These include mitoribosomal proteins and various RNA processing, modification and degradation enzymes. RNA crosslinking has been successfully applied to identify whole-cell poly(A) RNA-binding proteomes, but this method has not been adapted to identify mitochondrial poly(A) RNA-binding proteomes. Here we developed and compared two related method  ...[more]

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