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TMT spectrometry unveils key M6a interactorsTMT spectrometry unveils key M6a interactors

ABSTRACT: The membrane glycoprotein M6a -together with proteolipid protein (PLP), DM20 and M6b- belongs to the tetraspan PLP family. M6a is a neuronal surface protein that promotes neuronal stem cell differentiation, migration, neurite and axonal outgrowth, filopodia/spine induction and synapse formation in primary neuronal cultures and non-neuronal cell lines. M6a has two extracellular loops (EC1 and EC2), four transmembrane domains, one intracellular loop and the N and C terminus facing the cell cytoplasm. However, the complete mechanism of action or proteins associated with it through its extracellular loops remained unknown. In previous work we provided strong evidence that M6aÃƒâ€šÃ‚Â´s extracellular loops widely contribute to its function. To asses this question, we designed and purified a chimera protein (M6a-loops as a bait protein) which was subjected to co-immunoprecipitation (Co-Ip) with rat hippocampi homogenates followed by TMT-MS. The TMT-MS and data analysis was done by the Proteomics Core Facility from the European Molecular Biology Laboratory (EMBL, Heidelberg, Germany).

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Brain

SUBMITTER: Camila Scorticati

LAB HEAD: Camila Scorticati

PROVIDER: PXD017347 | Pride | 2020-06-29

REPOSITORIES: pride

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Source:

			Action	DRS
	Bert_181030_P0784_MR_CS_IP_1out10_R1.raw	Raw
	Bert_181109_P0784_MR_CS_IP_TMT_fr1.raw	Raw
	Bert_181109_P0784_MR_CS_IP_TMT_fr2.raw	Raw
	Bert_181109_P0784_MR_CS_IP_TMT_fr3.raw	Raw
	F075212.dat	Other

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Project description:Proteomics, the temporal study of proteins expressed by an organism, is a powerful technique that can reveal how organisms respond to biological perturbations, such as disease and environmental stress. Yet, the use of proteomics for addressing ecological questions has been limited, partly due to inadequate protocols for the sampling and preparation of animal tissues from the field. Although RNAlater is an ideal alternative to freezing for tissue preservation in transcriptomics studies, its suitability for the field could be more broadly examined. Moreover, existing protocols require samples to be preserved immediately to maintain protein integrity, yet the effects of delays in preservation on proteomic analyses have not been thoroughly tested. Hence, we optimised a proteomic workflow for wild-caught samples. First, we conducted a preliminary in-lab test using SDS-PAGE analysis on aquaria-reared Octopus berrima confirming that RNAlater can effectively preserve proteins up to 6 h after incubation, supporting its use in the field. Subsequently, we collected arm tips from wild-caught Octopus berrima and preserved them in homemade RNAlater immediately, 3 h, and 6 h after euthanasia. Processed tissue samples were analysed by liquid chromatography tandem mass spectrometry to ascertain protein differences between time delay in tissue preservation, as well as the influence of sex, tissue type, and tissue homogenisation methods. Over 3500 proteins were identified from all tissues, with bioinformatic analysis revealing protein abundances were largely consistent regardless of sample treatment. However, nearly 10 % additional proteins were detected from tissues homogenised with metal beads compared to liquid nitrogen methods, indicating the beads were more efficient at extracting proteins. Our optimised workflow demonstrates that sampling non-model organisms from remote field sites is achievable and can facilitate extensive proteomic coverage without compromising protein integrity.

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TMT spectrometry unveils key M6a interactorsTMT spectrometry unveils key M6a interactors

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