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Identification of proteins precipitated by means of Acyl-RAC

ABSTRACT: The objective was to perform protein identification by ESI-LC-MSMS on a sample subjected to Acyl-RAC assay in order to study protein palmitoylation. The user transfected human paxillin (mCherry-paxillin-CAIL) into a mouse cell line and performed Acyl-RAC assay. The aim of this analysis was to determine whether human paxillin is palmitoylated and confirm the quality of the Acyl-RAC technique to detect palmitoylated proteins.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Fibroblast

SUBMITTER: Bernhard Wehrle-Haller

LAB HEAD: Bernhard Wehrle-Haller

PROVIDER: PXD023810 | Pride | 2021-03-30

REPOSITORIES: Pride

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			Action	DRS
	20200929_QEHF25CM60_pl20093_MRipamonti_1.raw	Raw
	20200929_QEHF25CM60_pl20093_MRipamonti_1__F012717_.mzid.gz	Mzid
	20200929_QEHF25CM60_pl20093_MRipamonti_1__F012717_.mzid_20200929_QEHF25CM60_pl20093_MRipamonti_1__F012717_.MGF	Mzid
	20200929_QEHF25CM62_pl20093_MRipamonti_2.raw	Raw
	20200929_QEHF25CM62_pl20093_MRipamonti_2__F012718_.mzid.gz	Mzid

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Project description:We report here the identification of substrates of the depalmitoylating enzyme PPT1 by quantitative mass spectrometry. We utilized a stringent two-step Acyl Resin-Assisted Capture (Acyl RAC) screen in which increased palmitoylation in vivo in PPT1 knockout is the first selection criteria for substrates. We validated hits by direct depalmitoylation with PPT1 to identify bona fide substrates. Most proteins identified are previously reported to be palmitoylated and the few known PPT1 substrates were validated. Significantly, the screen identified >100 proteins not previously known to be depalmitoylated by PPT1, including a wide range of endocytic, signaling, synaptic adhesion, mitochondrial, and lysosomal proteins. These proteins collectively explain the many facets of CLN1 disease caused by the loss of PPT1 function. We determined that an Ig domain in synaptic adhesion molecules may be a PPT1 recognition site for depalmitoylation. Finally, we provide evidence that PPT1 participates in neuronal activity-dependent palmitoylation-depalmitoylation cycles at the synapse.

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