Dataset Information

Cell surface mapping and monitoring of cell surface dynamics

ABSTRACT: Here, we present a simple and robust N-linked glycoproteome enrichment protocol optimized for deep coverage of the plasma membrane proteome. The procedure was applied to characterize the cell surface proteome of 15 standard laboratory human cell lines and three primary cell types. We observed significant differences in receptor diversity and transporter abundances between primary cells and corresponding cell lines. Relative quantification using isobaric mass tagging enabled quantitative monitoring of dynamic processes on the cell surface in multiplexed experiments. We for the first time report a comprehensive analysis of the profound remodeling of the plasma membrane proteome during monocyte to macrophage differentiation of THP-1 cells. Treatment of these cells with the kinase inhibitor dasatinib (Sprycel) during differentiation severely compromised macrophage differentiation due to an off-target activity resulting in substantial morphological and functional changes and the loss of many macrophage-associated proteins.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): B Cell, T Cell, Monocyte, Permanent Cell Line Cell, Cell Culture, Macrophage

SUBMITTER: Maria Faelth Savitski

LAB HEAD: Christian Eberl

PROVIDER: PXD005846 | Pride | 2017-03-27

REPOSITORIES: Pride

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Publications

Monitoring Cell-surface <i>N</i>-Glycoproteome Dynamics by Quantitative Proteomics Reveals Mechanistic Insights into Macrophage Differentiation.

Kalxdorf Mathias M Gade Stephan S Eberl H Christian HC Bantscheff Marcus M

Molecular & cellular proteomics : MCP 20170323 5

The plasma membrane proteome plays a crucial role in inter- and intracellular signaling, cell survival, and cell identity. As such, it is a prominent target for pharmacological intervention. The relatively low abundance of this subproteome in conjunction with challenging extractability and solubility still hampers its comprehensive analysis. Here, we combined a chemical glycoprotein-tagging strategy with mass spectrometry to enable comprehensive analysis of the cell-surface glycoproteome. To ben ...[more]

PMID: 28336715

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Project description:In eukaryotes, regulation of mRNA translation enables a fast, localized and finely tuned expression of gene products. Within the translation process, the first stage of translation initiation is most rigorously modulated by the actions of eukaryotic initiation factors (eIFs) and their associated proteins. These 11 eIFs catalyze the joining of the tRNA, mRNA and rRNA into a functional translation complex. Their activity is influenced by a wide variety of extra- and intracellular signals, ranging from global, such as hormone signaling and unfolded proteins, to specific, such as single amino acid imbalance and iron deficiency. Their action is correspondingly comprehensive, in increasing or decreasing recruitment and translation of most cellular mRNAs, and specialized, in targeting translation of mRNAs with regulatory features such as a 5’ terminal oligopyrimidine tract (TOP), upstream open reading frames (uORFs), or an internal ribosomal entry site (IRES). In mammals, two major pathways are linked to targeted mRNA translation. The target of rapamycin (TOR) kinase induces translation of TOP and perhaps other subsets of mRNAs, whereas a family of eIF2 kinases does so with mRNAs containing uORFs or an IRES. TOR targets translation of mRNAs that code for proteins involved in translation, an action compatible with its widely accepted role in regulating cellular growth. The four members of the eIF2 kinase family increase translation of mRNAs coding for stress response proteins such as transcription factors and chaperones. Though all four kinases act on one main substrate, eIF2, published literature demonstrates both common and unique effects by each kinase in response to its specific activating stress. This suggests that the activated eIF2 kinases regulate the translation of both a global and a specific set of mRNAs. Up to now, few studies have attempted to test such a hypothesis; none has been done in mammals. We use array analysis to determine the global mRNA shift into polysomes following a stress response, and to compare the translational response following activation of GCN2 versus PERK, two of the four eIF2alpha kinases. This SuperSeries is composed of the following subset Series:; GSE11496: Expression data from Gcn2 wild-type and knockout mouse liver perfused with or without methionine; GSE11684: Expression data from Perk wild-type and knockout mouse liver perfused without or with 2,5-di-tert-butylhydroquinone Experiment Overall Design: Refer to individual Series

Project description:In eukaryotes, regulation of mRNA translation enables a fast, localized and finely tuned expression of gene products. Within the translation process, the first stage of translation initiation is most rigorously modulated by the actions of eukaryotic initiation factors (eIFs) and their associated proteins. These 11 eIFs catalyze the joining of the tRNA, mRNA and rRNA into a functional translation complex. Their activity is influenced by a wide variety of extra- and intracellular signals, ranging from global, such as hormone signaling and unfolded proteins, to specific, such as single amino acid imbalance and iron deficiency. Their action is correspondingly comprehensive, in increasing or decreasing recruitment and translation of most cellular mRNAs, and specialized, in targeting translation of mRNAs with regulatory features such as a 5’ terminal oligopyrimidine tract (TOP), upstream open reading frames (uORFs), or an internal ribosomal entry site (IRES). In mammals, two major pathways are linked to targeted mRNA translation. The target of rapamycin (TOR) kinase induces translation of TOP and perhaps other subsets of mRNAs, whereas a family of eIF2 kinases does so with mRNAs containing uORFs or an IRES. TOR targets translation of mRNAs that code for proteins involved in translation, an action compatible with its widely accepted role in regulating cellular growth. The four members of the eIF2 kinase family increase translation of mRNAs coding for stress response proteins such as transcription factors and chaperones. Though all four kinases act on one main substrate, eIF2, published literature demonstrates both common and unique effects by each kinase in response to its specific activating stress. This suggests that the activated eIF2 kinases regulate the translation of both a global and a specific set of mRNAs. Up to now, few studies have attempted to test such a hypothesis; none has been done in mammals. We use array analysis to determine the global mRNA shift into polysomes following a stress response, and to compare the translational response following activation of GCN2 versus PERK, two of the four eIF2alpha kinases. Experiment Overall Design: Gcn2 wild-type or knockout mouse liver were perfused with complete amino acids media or media lacking methionine for RNA extraction and hybridization of Affymetrix microarrays. RNA was extracted from unfractionated liver samples and polysome fraction of samples separated on sucrose density gradient. To minimize biological variations, we pooled RNA from two perfused liver samples to use in each array analysis. The conditions were total and polysome fraction of Gcn2+/+, +Met or -Met; total and polysome fraction of Gcn2-/-, +Met or -Met. Each array analysis was done in duplicate.

Dataset Information

Cell surface mapping and monitoring of cell surface dynamics

Publications

Monitoring Cell-surface <i>N</i>-Glycoproteome Dynamics by Quantitative Proteomics Reveals Mechanistic Insights into Macrophage Differentiation.

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