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New insights in osteogenic differentiation revealed by mass spectrometric assessment of phosphorylated substrates in murine skin mesenchymal cells

Erik Halcsik1, Maria Fernanda Forni1, Andre Fujita2, Thiago Verano-Braga3, Ole Nørregaard Jensen3 and Mari Cleide Sogayar1*

Author Affiliations

1 Chemistry Institute, Department of Biochemistry, Cell and Molecular Therapy Center (NUCEL/NETCEM), School of Medicine, University of São Paulo, São Paulo 05508-000, SP, Brazil

2 Department of Computer Science, Institute of Mathematics and Statistics, University of São Paulo, Rua do Matão 1010, São Paulo, SP 05508-090, Brazil

3 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

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BMC Cell Biology 2013, 14:47  doi:10.1186/1471-2121-14-47

Published: 22 October 2013

Additional files

Additional file 1:

Phosphorylated peptides found in MS experiments. Acession number, delta score, retention area (for light medium and heavy labeled peptides) of phosphorylated peptides.

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Additional file 2:

Phosphorylated proteins found and relative ratio variation for different timepoints of BMP2-induction and calculated p-value for all peptides corresponding to a specific protein. For a given accession number corresponding to a protein, a subset of peptides were found in MS experiments, and the ratio between each timepoint was calculated using StatQuant software according to the area of extracted chromatogram. Statistical analysis was calculated for a population of peptides corresponding to a same protein accession.

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Additional file 3:

Phosphorylated sites found in peptides according to MS experiments and kinase phosphorylation analysis using NetworKIN 2.0 Beta (http://www.networkin.info/version_2_0/ webcite). To investigate which kinases could be involved in the phosphorylation of peptides found in MS experiments, NetworKIN 2.0 Beta kinase databank was used to match phoshorylated serine, threonine and tyrosine in peptides found to be phoshorylated, according to prior experiments in literature for a given kinase based on PhosphoELM (http://phospho.elm.eu.org/ webcite) and on Phosphosite (http://www.phosphosite.org webcite).

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Additional file 4:

Network of proteins which were found to interact with phosphorylated proteins found. The list of phosphoproteins found were subjected to Ingenuity Pathway Analysis (IPA) to investigate probable protein interactions for each cellular compartment. Proteins described to be transcription factors were selected to investigate the activation of osteoblast related genes by quantitative real-time PCR.

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Additional file 5:

Predicted network of interaction for phosphoproteins found using Ingenuity Pathway Analysis. Ingenuity Pathway Analysis was used to identify the network of proteins which could interact with the phosphoproteins identified. Dashed arrows represent the predicted interactions and the full arrows represent a confirmed interaction. Proteins were separated by cell compartment and proteins known to be transcription factors were selected to further analysis to investigate possible activators of osteoblast differentiation by real-time quantitative PCR.

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Additional file 6:

Motifs and Pre-selected genes used in RT-PCR experiments. The motifs listed were used to search for correspondents in the promoter region of the pre-selected genes. Those which displayed at least one match matches for the sequences (SP1, c-Myc e NF-ƙB) were selected for the qRT-PCR analysis.

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