PXD012707

PXD012707 is an original dataset announced via ProteomeXchange.

Title	Local translation of mitochondrial proteins at the synapse
Description	To systematically explore the dynamics of the synaptic proteome upon neuronal stimulation we employed a simple model system which is based on isolated synaptoeurosomes (SN). Synaptoneurosomes are preparations freshly obtained from the brain and enriched in synapses, containing both pre and postsynaptic compartments. After the isolation they were in vitro stimulated. Protocol was set to induce NMDARs on the synaptoneurosomes which initiate calcium signaling in neurons and lead to long lasting responses such as long term potentiation (LTP). The control experiments in which we incubated SN with RNAse and proteinase K have shown that the translation is taking place inside the SN vesicles. Having established that translation in synaptoeurosomes prepared by us relay on endogenous mRNA we assumed that all proteins which abundance is increased after in vitro stimulation must be locally synthetized on base of mRNA already transported into synapses. In order to identify newly synthetized proteins we in vitro stimulated synaotoneurosomes and combined it with high-resolution quantitative MS techniques (Figure 1A). It is well established that quantitative mass spectrometry heavily depends on physico-chemical properties of peptides and dynamic range (~4 orders of magnitude) of sample which limits a depth of analysis. Some of tryptic peptides would be better detectable unmodified, other when modified for example with stable isobaric labeling techniques iTRAQ or TMT tag. We begin with label-free experiments, but it was mainly limited by dynamic range of protein in SN, we overcome that issue using iTRAQ 8-plex (4 x controls, 4 x stimulation) coupled with sample fractionation prior to LC-MS.. For this reason we used also TMT 10 plex, which allows us to measure 5 control and 5 stimuli samples at once. Using all three techniques we quantified (FDR 0.01) 2187 proteins. Number of identified proteins could be considered as relatively low for shotgun MS experiment. But it should be noticed that synaptical proteome is way different then “normal” cellular proteome since is depleted of nuclear, many of cytoplasmic and other organelles proteins. Finally some of proteins could be heavily modified. . Importantly, we identified 439 of which were significantly (Whitney-Mann p<0.05) upregulated in synaptoneurosomes in response to the stimulation which corresponds to 20% of identified proteins. The average fold change was relatively low which was however expected because it is not possible to quantitatively change a proteome of synapse through short pule of in vitro stimulation. But four replicates are often not enough for reliable statistic calculation of protein level changes. That is because of low level of proteins that could be synthetase at the synapse, due to its low ATP/syntethic potential. Which results in changes on average 3% of protein level.
HostingRepository	PRIDE
AnnounceDate	2020-08-12
AnnouncementXML	Submission_2020-08-12_00:30:08.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Dominik Cysewski
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	phosphorylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2019-02-14 02:30:54	ID requested
⏵ 1	2020-08-12 00:30:09	announced

Kuzniewska B, Cysewski D, Wasilewski M, Sakowska P, Milek J, Kulinski TM, Winiarski M, Kozielewicz P, Knapska E, Dadlez M, Chacinska A, Dziembowski A, Dziembowska M, Mitochondrial protein biogenesis in the synapse is supported by local translation. EMBO Rep, 21(8):e48882(2020) [pubmed]

curator keyword: Biological

submitter keyword: synaptosomes, local translation, quantitative proteomics

Magdalena Dziembowska
contact affiliation	Laboratory of Molecular Basis of Synaptic Plasticity Centre of New Technologies, University of Warsaw
contact email	m.dziembowska@cent.uw.edu.pl
lab head
Dominik Cysewski
contact affiliation	Institute of Biochemistry and Biophysics Polish Academy of Sciences
contact email	dominikcysewski@gmail.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD012707
     1. Label: PRIDE project
     2. Name: Local translation of mitochondrial proteins at the synapse