Dataset Information

ITRAQ MS of mouse LbetaT2 cells transfected with Galphas siRNA vs. scrambled siRNA

ABSTRACT: Twenty million LbetaT2 cells were transfected with either control or Galphas siRNA, then were seeded in 100-mm cell culture plates in DMEM + 10% FBS. Two days later, cells were washed twice with pre-warmed PBS. Conditioned media was harvested another 24 h later, and centrifuged at 20,000 g for 10 min at 4°C to remove cell debris. To enrich secreted proteins in the conditioned media, conditioned media samples were centrifuged using Amicon centrifugal filters with a 3kDa cutoff (Millipore, Billerica, MA). A total of 8 concentrated conditioned media samples were independently prepared: 4 samples from control siRNA-treated cells, and 4 samples from Galphas siRNA-treated cells. Samples were stored at –70°C until they were sent to the Mount Sinai Proteomics Core Facility.HPLC-isobaric tags for relative and absolute quantitation mass-spectrometry (iTRAQ MS) - Data analysis ProteinPilot 3.0 (AB Sciex) was used to search the MS/MS spectra for protein identification and quantitation with its searching algorithm Paragon 3.0.0.0 (*Reference). The protein database used for searching was Uniprot mouse fasta file (release-2010_11). The search parameters include quantitation for iTRAQ 8-plex (peptide-labeled), MMTS for cysteine alkylation, trypsin for enzyme digestion, biological modifications for ID focus, and taxonomy set for Mus musculus. The detected protein threshold was set to 1.3 (95% confidence). Additionally, we converted our AB Sciex mass spectral data (TOF/TOF data) into an mzML format, using the AB Sciex MS Data Converter (beta version 1.3) tool. Finally, we used the command line tool group2xml, which is included with ProteinPilot Software, to convert the .group search engine result file to an XML file.

OTHER RELATED OMICS DATASETS IN: PRJNA229506

INSTRUMENT(S): 4800 Proteomics Analyzer

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Hanna Pincas

LAB HEAD: Stuart C. Sealfon

PROVIDER: PXD000063 | Pride | 2016-09-27

REPOSITORIES: Pride

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Publications

Characterization of Gonadotrope Secretoproteome Identifies Neurosecretory Protein VGF-derived Peptide Suppression of Follicle-stimulating Hormone Gene Expression.

Choi Soon Gang SG Wang Qian Q Jia Jingjing J Chikina Maria M Pincas Hanna H Dolios Georgia G Sasaki Kazuki K Wang Rong R Minamino Naoto N Salton Stephen R J SR Sealfon Stuart C SC

The Journal of biological chemistry 20160727 40

Reproductive function is controlled by the pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH), which regulates the expression of the gonadotropins luteinizing hormone and FSH in pituitary gonadotropes. Paradoxically, Fshb gene expression is maximally induced at lower frequency GnRH pulses, which provide a very low average concentration of GnRH stimulation. We studied the role of secreted factors in modulating gonadotropin gene expression. Inhibition of secretion specifically ...[more]

PMID: 27466366

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Project description:Mutations in the RMRP gene are the origin of cartilage-hair hypoplasia. Cartilage-hair hypoplasia is associated with severe dwarfism caused by impaired skeletal development. However, it is not clear why mutations in the RMRP gene lead to skeletal dysplasia. Viperin is a known substrate of RMRP. Since chondrogenic differentiation of the growth plate is required for development of the long bones, we hypothesized that viperin functions as a chondrogenic regulator downstream of RMRP. Viperin protein is expressed throughout the stages of chondrogenic differentiation in vivo. Viperin gene expression is increased during knockdown of Rmrp RNA in the ATDC5 model for chondrogenic differentiation. Viperin is expressed during ATDC5 chondrogenic differentiation. Viperin knockdown reduces, while viperin overexpression increases overall protein secretion, with CXCL10 identified as a potential target via mass spectrometry-proteomics. CXCL10 protein expression is reduced during knockdown and increased during overexpression of viperin and CXCL10 protein expression coincides with viperin expression in ATDC5 chondrogenic differentiation. Viperin knockdown induces, while viperin overexpression reduces TGFβ activity. Furthermore, viperin knockdown conditioned media increases, while viperin overexpression conditioned media reduces chondrogenic differentiation of ATDC5 cells. TGFβ target genes Pai1 and Smad7 are increased during knockdown and reduced during overexpression of viperin. Moreover, TGFβ activity is reduced when differentiating ATDC5 cells are exposed to CXCL10 and, acting as a viperin overexpression mimic, CXCL10 similarly reduces chondrogenic differentiation of ATDC5. Lastly, we show that in CHH patient cells, RMRP expression is reduced and viperin expression is increased, coinciding with reduced chondrogenic differentiation and increased CXCL10 expression, possibly explaining the CHH phenotype. Together our data show that viperin may play a pivotal role in chondrogenic differentiation, with potential consequences for cartilage-hair hypoplasia pathobiology.

Dataset Information

ITRAQ MS of mouse LbetaT2 cells transfected with Galphas siRNA vs. scrambled siRNA

Publications

Characterization of Gonadotrope Secretoproteome Identifies Neurosecretory Protein VGF-derived Peptide Suppression of Follicle-stimulating Hormone Gene Expression.

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