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Targeted cross-linker delivery for in situ mapping of protein conformations and interactions in mitochondria

ABSTRACT: Current methods for intracellular protein analysis mostly require the separation of specific organelles or the change of intracellular environment. However, the functions of these proteins are determined by their native microenvironment as they usually form complexes with ions, nucleic acids, and other proteins. Thus, we have explored a method for in situ crosslinking and mapping of mitochondrial proteins in living cells. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are functionalized with dihexadecyldimethylammonium bromide (DDAB) to deliver protein crosslinkers into mitochondria. In situ crosslinked proteins are analyzed using mass spectrometry.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Yuwan Chen

LAB HEAD: Lihua Zhang

PROVIDER: PXD035433 | Pride | 2023-05-26

REPOSITORIES: Pride

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			Action	DRS
	20200906-in-vivo-cellexclude-2.raw	Raw
	20200906-in-vivo-cellexclude-2_200907005638.raw	Raw
	20200906-in-vivo-cellexclude-2_200907034615.raw	Raw
	20200906-juri-0225mMDSS-exclude-2-1.raw	Raw
	20200906-juri-0225mMDSS-exclude-2-2.raw	Raw

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Targeted cross-linker delivery for in situ mapping of protein conformations and interactions in mitochondria

Project description:Current methods for intracellular protein analysis mostly require the separation of specific organelles or the change of intracellular environment. However, the functions of these proteins are determined by their native microenvironment as they usually form complexes with ions, nucleic acids, and other proteins. Thus, we have explored a method for in situ crosslinking and mapping of mitochondrial proteins in living cells. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are functionalized with dihexadecyldimethylammonium bromide (DDAB) to deliver protein crosslinkers into mitochondria. In situ crosslinked proteins are analyzed using mass spectrometry. With this method, we have totally identified a total of 74 pairs of74 pairs of protein-protein interactions that do not exist in the STRING database. Our data on mitochondrial respiratory chain proteins (~93%) are also consistent with the experimental or predicted structural analysis of these proteins. Thus, we have established a promising technology platform for in situ defined protein analysis in cellular organelles under their native microenvironment.

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