Dataset Information

Stable isotope dynamic labelling of secretomes C4PR_LIV

ABSTRACT: We improve on currently-available resources by describing a mass spectrometry (MS)-based strategy using stable isotope dynamic labelling of secretomes (SIDLS) that discriminates between authentic secretory proteins and intracellular proteins within the secretome of cultured cells. By monitoring the rate of incorporation of labelled amino acids into newly synthesised proteins as they appear in the media, we can differentiate those proteins that have been destined for secretion, and exhibit rapid labelling, from those with low rates of labelling or low turnover relative to the growth rate of the cells which is a feature of intracellular proteins.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte, Esophagus Mucosa, Esophagus

DISEASE(S): Esophageal Cancer

SUBMITTER: Dean Hammond

LAB HEAD: Dean Hammond

PROVIDER: PXD007231 | Pride | 2019-10-08

REPOSITORIES: Pride

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Publications

Stable Isotope Dynamic Labeling of Secretomes (SIDLS) Identifies Authentic Secretory Proteins Released by Cancer and Stromal Cells.

Hammond Dean E DE Kumar J Dinesh JD Raymond Lorna L Simpson Deborah M DM Beynon Robert J RJ Dockray Graham J GJ Varro Andrea A

Molecular & cellular proteomics : MCP 20180618 9

Analysis of secretomes critically underpins the capacity to understand the mechanisms determining interactions between cells and between cells and their environment. In the context of cancer cell micro-environments, the relevant interactions are recognized to be an important determinant of tumor progression. Global proteomic analyses of secretomes are often performed at a single time point and frequently identify both classical secreted proteins (possessing an N-terminal signal sequence), as wel ...[more]

PMID: 29915148

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Project description:The subcellular localization of specific mRNAs is an evolutionary conserved mechanism that underlies the establishment of cellular polarity and specialized cell functions. In neurons, mRNA trafficking and local protein translation in dendrites provides an important mechanism that mediates synaptic development and plasticity. The significance of mRNA targeting and protein synthesis in axons, however, is still unclear. Only a small number of transcripts have been identified in axons to date, and their contribution to axon growth and neuronal survival remains largely unknown. Here, we report the results of a novel screen that allowed the separate identification of mRNAs localized in cell bodies and in axons of developing neurons. Using compartmentalized cultures of sympathetic neurons and Sequential Analysis of Gene Expression (SAGE), the screen identified more than 200 axonal mRNAs, including ones that encode cytoskeletal proteins and proteins that function in neural development and signal transduction. Importantly, several classes of transcripts were selectively enriched in axons, indicating that an active process drives the targeting of specific mRNAs from the cell bodies to the axons. This study is the first comprehensive and unbiased analysis of mRNA localization in subcellular domains of any neuronal cell type. We used compartmentalized chambers to culture neonatal rat sympathetic neurons (Campenot, 1977). In these cultures, the cell bodies are separated from the distal axons by a 1 mm wide Teflon divider, which maintains the cell bodies and axon terminals in separate fluid compartments. Primary rat sympathetic neurons are especially suitable for compartmentalized culture because they can be grown as a highly homogeneous population without glial cells. Neurons were seeded in the central compartment with nerve growth factor (NGF), and after a few days, the NGF was lowered in this compartment and supplied only to the peripheral compartment to stimulate axon growth. The anti-mitotic agent cytosine arabinoside C (Ara-C) was added to both compartments to remove non-neuronal cells. mRNA was then isolated after 12 days in culture (DIV) from cell body or axon compartments. As the initial mRNA content in axons was not sufficient to perform the SAGE analysis, both axon and cell body mRNAs were subjected to two rounds of linear amplification to obtain antisense RNA (aRNA). The amplified aRNA was then reverse transcribed and second strand synthesis was performed to proceed with the SAGE assay using the LongSAGE kit (Invitrogen) according to the manufacturer’s protocol.

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