BACKGROUND
fractures and bone loss impose high costs for the public healthcare system.
furthermore, delayed-healing fractures lead to recurrence lesion, with quality of life’s loss and increased patient morbidity.
in the normal healing process, the bone tissue function is regenerated through endochondral ossification and intramembranous ossification, which often occur at same time at the lesion site, under the influence of inflammatory agents, such as il <dig>  il <dig> and tnf-α  <cit> , which induce migration and proliferation of periosteum mesenchymal stem cells.
these cells differentiate into osteoblasts, the major step in the regenerative process.
however, during the individual’s lifetime, both the availability and the ability of these cells to differentiate diminish, leading to incomplete or total absence of tissue regeneration at the fracture site.
although physiological details are well understood, the molecular aspects of the differentiation process occurring in the osteoblast lineage from adjacent mesenchymal cells remain unclear.
to address this issue, autologous mesenchymal stem cells  have been utilized, improving the bone tissue regeneration capability and leading to reduction of both total costs and hospitalization period, with a significant decrease in lesion recurrence  <cit> .
these cells gained importance in regenerative medicine, due to their ability to differentiate into chondrocytes, adipocytes and osteoblasts, and facility with which they may be isolated from several organs, among which is the skin.
due to its function of protecting from exposure to deleterious agents, such as uv light, physical injuries and pathogens, the skin displays a high cell proliferation rate, which is maintained by the self-renewal and differentiation capabilities of the several stem cell populations present in skin niches  <cit> .
these cells are of particular interest, since they may be easily isolated from the skin, in reasonable amounts, being highly suitable for bone healing and repair  <cit> .
although it is known that osteogenic differentiation in mscs is initiated through activation of canonical pathways such as smad  proteins, the possible protein interactions with other pathways which may influence cell differentiation remain elusive.
the activation of different downstream signaling cascade pathways, includes hedgehog, wnt, pthr-p and bmps, which, in turn, activate the main transcription factors related to osteogenesis through their respective pathways  <cit> .
smads, for example, may be positively or negatively regulated by phosphorylation of different residues, leading to activation or suppression of the bmp-initiated signal  <cit> .
these kinase pathways, in turn, activate downstream effectors in the cytoplasm and nucleus by phosphorylating a network of substracts.
since the study of protein phosphorylation depends mainly on phosphospecific antibodies and the utilization of radioisotopes, identification of novel phosphorylation sites has been a laborious task.
however, the development of mass spectrometry techniques by detection of inorganic phosphate neutral loss through cid , originated from phosphoserine, phosphotyrosine and phosphothreonine, has made it possible to describe these novel phosphorylation sites in several proteins  <cit> .
even though the analysis of fragmented peptides using ms tools is an alternative method, due to their negative charge and low abundance, phosphorylated peptides display poor ionization and are subjected to signal suppression, when compared to the regular, non-modified peptides  <cit> .
therefore, it is necessary to enrich for the phosphorylated peptides population present in the sample and to eliminate interfering ions.
this may be accomplished by using a metal affinity chromatography, such as imac or tio <dig>  thus improving the detection levels of modified peptides  <cit> .
this technique, coupled with stable isotope labeling of peptides for quantitative proteomics, may provide information on the proteins which are differentially phosphorylated during bmp2-induced osteodifferentiation.
an unexpensive and practical method for quantitative proteomics is the use of stable-isotope dimethyl labeling  <cit> .
primary amine of tryptic peptides and the lysine ϵ-amino group can react with formaldehyde in the presence of cyanoborohydride through reductive amination, giving rise to dimethylated amine as the product.
depending on which stable isotope is used , different shifts in molecular mass may be achieved.
using both non-modified formaldehyde and cyanoborohydride, the mass shift is of 28 da.
using both deuterated formaldehyde and cyanoborohydride, the mass shift is of 32 da, and using 13c plus deuterated formaldehyde and cyanoborohydride, the mass shift is of 36 da.
each isotope differs from each other by 4 da per primary amine labeled, with the comparison between different samples being made by ms precursor ion identification on extracted chromatograms  <cit> .
here, we employed mass spectrometry coupled to tio <dig> metal affinity chromatography techniques to uncover new players involved in mouse skin mesenchymal cells  osteogenic differentiation.
RESULTS
quantitative phosphoproteome and proteome of msmsc cells subjected to rhbmp <dig> osteoblastic differentiation
msmsc cells cultured in 100 mm dishes were treated with rhbmp <dig> for different periods of time, in order to assess protein phosphorylation changes during the first steps of osteoblastic differentiation.
previous experiments using the osteoblast differentiation medium  showed intense calcification of our murine skin mscs in  <dig> and 21 days .
homogeneity of the skin dermal mscs was probed through a complete characterization of cd markers, namely: cd <dig>  cd <dig>  cd <dig>  cd <dig> and cd <dig> , utilizing only cell populations displaying greater than 90% purity for the osteogenic differentiation assays.
due to the use of three different isotopes to label the samples and five different timepoints, it was necessary to carry out two independent experiments, each of which containing a light, an intermediate and a heavy isotope .
we also evaluated protein level changes, through analysis of the total protein fraction  in order to compare total protein levels alterations.
since the tio <dig> flowthrough and wash fractions represent more than 70% of the sample  <cit>  and are highly complex, another fractionation step was performed.
hilic separation was used to reduce sample complexity, according to protein hydrophilicity.
data analysis
the “raw” data acquired from thermo ltq-xl orbitrap® was converted to “.mgf” files and an in-house mascot server was used to search for peptides containing dimethyl and carbamylation as a fixed modification and for phosphorylation in serine, tyrosine and threonine.
the thermo proteome discoverer software, version  <dig>  was used to quantify all peptides based on the total area of extracted chromatogram , and the absolute values were normalized using a lowess  algorithm .
these data were input into the statquant software to evaluate the overall protein ratio by calculating the mean peptide ratio for all peptides corresponding to a given protein.
the list for all peptides and phosphopeptides quantified can be accessed in the additional file  <dig>  and a summary of upregulated and downregulated phosphoproteins in each experiment, sorted by period of time indutction with rhbmp <dig> is shown in additional file  <dig>
phosphosite localization
to assign phosphorylation sites, normalized mascot delta score  was used  <cit> .
mascot delta score is the difference between the top two scores for the peptides identified by a given spectrum.
dividing this value by the score of the top score  peptide, normalized delta score is obtained.
in order to have 1% flr  for correct phosphosite assignment with 99% certainty, peptides with nmd-score below  <dig>  were discarded .
a total of  <dig> unique phosphosites with 99% certainty that the sites were assigned correctly  were identified.
these sites were found on  <dig> different proteins and their distributions were  <dig> %,  <dig> % and  <dig> % for ps, pt and py, respectively, which is comparable to previous works for mammalian cell types.
all validated phosphosites with their md-scores are listed in additional file  <dig>
phosphorylation motif database search
the analysis carried out to determine which kinase could possibly be involved in phosphsorylation of a given phosphorylated residue from phosphoproteome data was performed using the networkin site .
figure  <dig> shows a summary of the complete dataset  represented by a graph containing kinase motifs occurrencies.
network analysis using the ingenuity pathway analysis software
in order to evaluate possible intracellular interactors with the phosphopeptides found, a network analysis was performed.
the ingenuity pathway analysis software  was used to map relationships among proteins, distributed into different cellular compartments.
from the total list of proteins found to interact with phosphoproteins , hits containing a transcription factor function were selected for further analysis of dna-binding motifs in osteoblast differentiation-related genes.
non-phosphorylated population of peptides were classified according to biological process using the gene ontology blast2go software  in order to map and annotate gene ontologies .
predicted network of interaction for phosphoproteins found using ingenuity pathway analysis.
the list of phosphoproteins found were subjected to ingenuity pathway analysis  to investigate problable protein interactions for each cellular compartment.
proteins described to be transcription factors were selected to investigate the activation of osteblast related genes by quantitative real-time pcr.
consistently upregulated proteins were input in blast2go software for mapping and annotation of gene ontologies.
biological processes over-represented were selected and classified according to level.
top: low levels; bottom.
analysis of the promoter region of osteoblast-related genes for the presence of responsive elements for the bmp2-regulated transcription factors
after obtaining the list of transcription factors for the ingenuity® network analysis, a curated database for transcription target genes, tred was used to find target genes and text mining was performed to find which target genes are related with osteoblastic differentiation.
we used the jaspar database  <cit>  which contains a curated, non-redundant set of profiles, derived from published collections of experimentally defined transcription factor binding sites for eukaryotes and sorted out the transcription factor which have well defined binding motifs   <cit> .
these motifs were used as a template for a search in the promoter region of the pre-selected genes, using the ensembl cisred database  <cit>  and those which displayed at least one match or multiple matches for the sequences were selected for the qrt-pcr analysis .
the consensus sequences of sp <dig>  c-myc and nfkb were selected among others because they were present in the promoter region in more them 80% of the selected genes for qpcr validation.
analysis of differentially expressed genes involved in osteoblastogenesis activated by bmp2-induced transcription factors
we used analysis of regulatory networks in order to investigate which transcription factors were activated, and which of them are related with activation of osteoblast-related genes .
thirteen genes were selected to evaluate their role in osteoblastic differentiation of msmsc cells, and to confirm the in silico analysis.
from the initial list of genes investigated, ten  were found to be upregulated at different timepoints .
the tgfb cytokine ant its receptor, tgfbr <dig>  displayed the regulated motifs in their promoter regions.
the mrna relative levels of these two genes were evaluated after 10 min, 30 min, 1 h and 2 h of exposure to rhbmp <dig>  the relative levels of tgfb <dig> were upregulated more than two times after 30 min of rhbmp <dig> induction , but after reaching this peak, the relative levels decreased to basal levels after 2 h. this pattern was followed by a subsequent increase in the tgfbr <dig> mrna relative levels of up to  <dig>  fold at 1 h and more than  <dig>  fold at 2 h .
since the synthesis of extracellular matrix compounds, such as collagens, is known to be regulated during osteo differentiation, we selected two members of the collagen family that displayed the selected motifs, namely: collagen  <dig> and 4a.
both ecm components were upregulated, with collagen  <dig> displaying a punctual increase at 1 h after stimulus  and collagen 4a followed a progressively rising pattern .
related to collagens and tgfb, the osteogenesis related gene twist presents a downregulation pattern  from the basal levels during the beginning of the differentiation and after that a slight increase  at 1 h, a decrease to  <dig>  fold at 2 h .
expression of smad <dig> was accessed, in order to stabilish a possible crosstalk between tgf-beta/activin signaling during bmp2-driven osteogenesis.
the mrna relative levels of smad <dig> were accessed, presenting a slight increase of  <dig>  fold at 10 min and a major increase of more than  <dig>  fold at 2 h .
list of primers used to amplify the cdna in order to investigate changes in mrna upon bmp <dig> treatment in qrt-pcr.
f: forward; r: reverse.
we also evaluated a set of four transcription factors which, in addition to presenting the regulated motifs in their promoter regions, were key elements during the osteoprogenitors differentiation.
the relative mrna levels of runx <dig> were the first to be upregulated, increasing almost  <dig> fold after 30 min, with a drastic descent to levels similar to basal levels after 1 h .
another important transcription factor, dlx- <dig>  displayed a progressive increase at 10 min  and 30 min  reaching a peak at 1 h , followed by a sharp decrease to basal levels at 2 h .
the transcription factor osterix displayed a stepwise increase, beginning at 10 min, and reaching up to  <dig> fold after 2 h of stimulation .
similarly, the sox <dig> mrna level was upregulated at 30 min  and 1 h  .
discussion
in the present study, we used murine skin mesenchymal cells  and stable dimethyl isotope labeling to quantify abundant proteins and phosphoproteins using tio <dig> metal affinity chromatography, coupled with mass spectrometry, at five different periods of rhbmp <dig> induction, namely:  <dig>   <dig>   <dig>   <dig> and 120 min.
from 150 μg of the combined samples, it was possible to identify and quantify  <dig> distinct phophoproteins and  <dig>  distinct proteins, in all replicates.
based on the data acquired, and, also, on references from the literature, we proposed a model for bmp2-mediated osteodifferentiation differentiation of these msmscs cells.
previous experiments carried out with these msmscs, subjected to the osteoblast differentiation medium  showed intense calcification at  <dig> and 21 days of treatment, with greater than 80% of the cells being alizarin red-positive” .
this experiment could not be carried out solely with bmps-supplemented culture medium, due to its lack of mineral components , which is necessary for mineralization.
the data found showed to be compatible with bone development, since bmps act at the very early stages of cells differentiation to the osteoblastic lineage, but, later on in the process, these cells incorporate mineral precursors and originate the calcified bone tissue.
the kinases which showed the highest number of phosphorylation motifs in phosphodata were represented, as well as gene activation for each time period studied .
we used triplex stable isotope dimethyl labeling to compare five different time periods of rhbmp2-induced osteoblastic differentiation of skin mesenchymal cells, combined into two different experimental groups .
this was necessary in order to correctly compare the phosphoprotein ratios with their respective protein levels, since we do not expect a wide protein level variation during the period studied and, also, to avoid aberrations in phosphoprotein variation.
similarly, song and colleagues  <cit>  used similar approach through a pseudo-triplex dimethyl labeling in liver tissue samples and hepatocellular carcinoma.
each pooled sample was split according to the affinity of peptides for the tio <dig> column.
the tio2-flowthrough fraction was subjected to hilic and fractionated.
since this step decreased the sample complexity, it enhanced the number of peptides which could be identified and quantified, when compared to the entire pool, which was directly injected into the lc/ms .
since we started from a relatively low amount of sample , no improvement in the number of detected peptides was obtained using hilic in phospho-enriched samples.
analysis of the data also showed that there is a tendency for many phosphopeptides to be upregulated between 30 min and 1 h of rhbmp <dig> treatment, correlating with the period of activation for the dlx <dig> transcription factors which trigger the expression of runx <dig> and osx, both of which are upregulated upon rhbmp <dig> administration  <cit> .
in order to compare measurements across lc-ms/ms experiments and to correct for non-biological variation , data normalization is a crucial step prior to any further analysis.
the standard normalization assumed in lc/ms experiments is based on dividing all peptide ratio values by log <dig>  however, notice that this procedure only divides the peptide abundance by a common factor, re-scaling the relative abundance of the peptide.
in other words, this “within sample” normalization does not remove the bias in the quantities across experiments.
in order to remove the systematic errors introduced in different experiments, we applied the lowess regression, a technique commonly applied to microarray data analysis  <cit> .
one premise to apply lowess normalization is that the differences among the overall intensity of different experiments would be the consequence of non-biological variation, i.e., most peptides will not show a significant change in the abundance between the two compared samples .
briefly, in a well performed experiment, the scatter-plot of peptides of one sample versus another would cluster the peptides along a straight line, and the slope would be equal to  <dig>  normalization of these data is equivalent to calculating the best-fit slope using regression techniques and adjusting the intensities so that the calculated slope is  <dig>  however, sometimes, the intensities may be non-linear, therefore, local regression techniques, such as lowess regression, are more suitable.
lowess regression  <cit>  is estimated through a locally weighted polynomial regression for a subset of peptides in the neighborhood of each peptide.
for more details, please refer to  <cit> .
bmp <dig> induces phosphorylation of substrates for different kinases in msmscs
kinase prediction analysis using the networkin database, from the phosphorylated peptides found, suggested that, three major kinases could be acting as effectors of phosphorylation upon bmp <dig> treatment, namely: casein kinase ii , p <dig> mapk and jnk.
these kinases are followed, to a lesser extent, by activin receptors and the cdk family of kinases.
these data are in agreement with the literature of the field, since bragdon and colleagues  <cit>  showed the involvement of ck <dig> in bmp2-induced cells.
the release of ck <dig> from bmp receptor type i  is related with osteblastogenesis, since specific peptides which interfere with this interaction, destabilize the ck2-bmpri complex and enhance osteoblastic differentiation.
it is possible that the role of ck <dig> in osteogenesis is much more than its release from bmpri, involving many of the substrates found in this work and even other ones which could contribute to the enrollment of these undifferentiated stem cells to osteoblastogenesis.
the involvement of p <dig> mapk in bmp2-driven osteoblatogenesis is well-known.
several studies  <cit>  show activation of p <dig> within the first hour of bmp <dig> induction, and activation of dlx <dig> and osx, essential genes involved in osteblastic differentiation  <cit> , as well as alkaline phosphatase.
we confirmed these data in our model using quantitative real-time pcr experiments, showing an increase in mrna relative expression for osx and dlx <dig>  it is interesting to note that p <dig> may be involved in phosphorylation of several phosphoproteins found in our study, since  <dig> sites were predicted to be phosphorylated by this kinase.
upon bmp <dig> treatment, jnk may also be activated, as previous studies described  <cit> .
we found that 9% of all sites could be phosphorylated by this kinase up to 2 h of bmp <dig> treatment.
interestingly, jnk is transiently activated in mc3t3-e1cells, in a short window , stimulating the expression of osteocalcin.
however, at late periods of bmp <dig> induction, jnk acts inhibiting the runx <dig> function by its phosphorylation at ser  <dig> in c2c <dig> cells  <cit> .
these results show the dual function of jnk in osteoblastogenesis, which is regulated in a time-dependent manner.
at early periods of time, jnk may have a role inducing osteogenesis, by phosphorylating intracellular substrates and augmenting the cellular sensibility for bmp <dig>  <cit> .
on the other hand, at late periods, jnk would participate by slowing down the intracellular signaling for osteodiffentiation.
similar number of phosphorylated sites were found for the cdk group of kinases.
these kinases are related with cell cycle progression, and their activation or inhibition is associated with proliferation and quiescence, respectively.
at a first glance, the activity of cdk kinases could lead to an impairment of osteoblastic differentiation, due to stimulation of cell proliferation.
the role of cdk in osteoblastic differentiation is not well understood yet, however, its inhibitor, the p <dig> protein, has been involved in osteoblastic differentiation since p <dig> null mice exhibit enhanced osteoblastic differentiation, and overexpression of p <dig> protein delays bone formation  <cit> .
it is possible that p <dig> could act independently of cdk, activating or repressing genes in the nucleus, with its role controlling osteoblastic differentiation being more complex than simply regulating the cell cycle.
further studies will be required to address this issue and how cdk/p <dig> regulation participates in osteoblastic differentiation.
biological processes overrepresented in bmp2-treated msmscs
the proteomic data obtained were analyzed using the gene ontology classification.
we observed which gene ontologies could be representative of the upregulated genes.
surprisingly, we found a high number of ontologies containing the following terms: multicellular organismal and anatomical structure development , signal transduction  signaling , cell differentiation , cell surface receptor linked signaling pathway  and phosphorylation  at the first hour of bmp <dig> treatment, in contrast with the first  <dig> and 30 min periods of induction, which showed a few gene ontologies with these terms assigned.
this can be due to the fact that short periods of time are not sufficient to change the overall amount of protein in the cell  <cit> , therefore, transcription and translation of new proteins must take place before we can observe changes in protein levels, which are sufficient to affect the gene ontologies classification observed.
nevertheless, comparing the second hour of bmp <dig> induction with the first one, less gene ontologies could be classified, leading to the conclusion that these proteins involved with signaling are regulated within the first hour bmp <dig> induction.
bmp2-treated msmscs phosphorylate intracellular messengers which, in turn, activate osteoblastic-related genes
bmp <dig> induction was shown to modify the post-translational modifications of intracelular proteins, at the timepoints studied.
in order to investigate how these phosphorylated proteins activate transcription factors, and whether they are related with the activation of osteoblastic genes, a network analysis of proteins found in the phosphoproteome of bmp2-treated msmscs was carried out.
through ingenuity network analysis, we found different transcription factors related with the phospho data.
however, not all of the transcription factors found were described to have any participation in osteoblast differentiation, or activation of osteoblastic related genes.
using a curated database for transcription target genes, tred, a transcription factors binding motifs occurrence, jaspar, and the literature on the field to search for osteoblastic target genes, one by one, we found three transcription factors from the ingenuity output list, displaying important roles in osteoblastogenesis, namely: sp <dig>  c-myc e nf-ƙb.
tgf-β/bmps are widely recognized for their role in bone formation during mammalian development, exhibiting versatile regulatory functions in the body  <cit> .
in accordance with this finding, we observed increased levels of the mrna for both the tgfb cytokine and for its receptor tgfbr.
also, signaling transduction by tgf-β/bmps occurs specifically through both canonical smad-dependent pathways  and a non-canonical smad-independent signaling pathway .
following tgf-β/bmp induction, both the smad and p <dig> mapk pathways converge at the runx <dig> gene to control mesenchymal precursor cell differentiation, which has also been found to have increased mrna levels.
sox <dig>  a transcription factor of the sex-determining region y -related high mobility group box family of proteins, is crucial for skeletal development and marks all osteoblastic progenitors  <cit> , being capable of inducing runx <dig> expression  <cit>  .
however, the role of sox <dig> in osteoblastic differentiation is not completely understood.
conditional deletion of sox <dig> in the limb bud mesenchyme led to the absence of chondrocytes and osteoblasts  <cit> .
contrastingly, when sox <dig> was deleted in the neural crest cells that contribute to the craniofacial skeleton, the cells which normally form chondrocytes expressed osteoblasts markers  <cit> , suggesting the existence of a the bipotential progenitor.
however, sox <dig> is not expressed by mature osteoblasts  <cit>  and this is the probable cause of its downregulation after 2 h of the stimulus.
col <dig> and col4a display functions related with the building of the basal membrane for the newly-formed mature bone tissue.
a recent report of comparative transcription of various fetal and adult mesenchymal stem cells sources through quantitative pcr profiling unveiled that collagens, such as collagen  <dig> and  <dig>  were upregulated during several types of osteogenic differentiation, such as the one reported in this manuscript with the levels of these two extracellular matrix components being increased  <cit> .
supporting these findings, it has been reported that site mutations in collagen  <dig> leads to high bone mass in osteogenesis imperfecta  <cit> .
since the bhlh transcription factor twist inhibits osteoblast differentiation through binding to a dna-binding domain in runx <dig>  <cit> , the early downregulation of this gene to levels below the basal level at  <dig> and 30 min could be indicative that the differentiation process was mediated by runx <dig>  moreover, it has been shown that runx <dig>  a runt domain-containing transcription factor, is indispensable for osteoblastic differentiation during both endochondral and intramembranous ossification and the function of mature osteoblasts, including the synthesis of bone matrix.
homozygous deletion of runx <dig> in mice resulted in a complete lack of osteoblasts  <cit> .
our results show a sustained increase in the mrna levels of this transcription factor after 30 min, pointining to the involvement of this gene in the osteogenesis induced by exposure to bmp <dig>  another essential gene related with osteoblastic differentiation is osx, a transcription factor containing three zinc-fingers  <cit> .
osx was discovered as a bmp-induced gene in c2c <dig> cells, with its deletion resulting in complete absence of osteoblasts in mouse embryos, despite the relatively normal expression of runx <dig>  <cit> , which indicates that osx is activated after runx <dig> during osteoblastic differentiation.
in accordance, we observed that after a significant increase in runx <dig> after 30 min of induction, a consistent increase of mrna osx levels is observed up to 2 h after stimulus.
finally, we analized the expression levels of dlx- <dig>  an homeobox gene that plays an essential role in craniofacial, axial, and appendicular skeletal development  <cit> , and specifically regulates runx <dig> expression by binding to the homeodomain-response elements in the runx <dig> distal promoter  <cit> .
the increased amounts of dlx- <dig> after exposure to bmp <dig> indicates that this gene is also present in our differentiation event, generating a reliable axis between dlx-5/runx-2/osx.
novel phosphorylated candidates found upon bmp <dig> treatment of msmscs
from all three independent experiments, we chose proteins which displayed increased phosphorylation upon bmp <dig> treatment: a group of proteins related with cytoskeletal rearrangement and ras protein signal transduction.
cytoskeletal rearrangement is observed during osteoblastic differentiation through the shift from a fibroblast-like to a spheric phenotype, upon induction with supplemented osteogenic differentiation medium , being antagonized by treatment with cytochalasin d, leading to a reduction of differentiation markers expression  <cit> .
thus, catenin alpha  <dig>  alpha parvin, septin- <dig>  caldesmon, microtubule associated proteins 1b and  <dig>  nexilin, cytoplasmic dynein  <dig> light intermediate chain and isoforms of lamin a/c and plectin  <dig> were found to be upregulated at all time periods studied.
together with the previous studies which had described activation of these proteins using odm  <cit> , we found that these proteins were also activated upon bmp <dig> treatment.
this may be explained by the fact that a common subset of proteins can be activated by both bmp <dig> and components of odm, phosphorylating other proteins related which cytoskeletal rearrangement.
another protein related with cytoskeletal rearrangement found in our experiments was rho gtpase-activating protein.
the rho family of gtpases plays an important role in osteoblastic differentiation, shown by differentiation to osteogenesis of constitutively rhoa-expressing mesenchymal stem cells  <cit> .
other proteins involving signaling pathways in osteoblastic differentiation were positively phosphorylated, namely: transforming growth factor beta  <dig> induced transcript  and bcl-2-associated transcription factor  <dig> displayed increasing phosphorylation levels.
these proteins are related to the wnt pathway and, specifically, hic- <dig> was involved in regulation of intracellular signals by smad  <dig>   <dig> and  <dig>  effector proteins of the canonical bmp <dig> signaling pathway  <cit> .
CONCLUSIONS
stable isotope dimethyl labeling of peptides may be used to quantify small amounts of proteins phosphorylated in cell extracts.
during bmp2-induced differentiation in skin derived mesenchymal stem cells, it was possible to acess different proteins, which many of them were found to be phosphorylated in different timepoints, giving new cues about the events that occur in the short term of osteoblastic differentiation.

 fractures and bone loss impose high costs for the public healthcare system. furthermore, delayed-healing fractures lead to recurrence lesion, with quality of life’s loss and increased patient morbidity. in the normal healing process, the bone tissue function is regenerated through endochondral ossification and intramembranous ossification, which often occur at same time at the lesion site, under the influence of inflammatory agents, such as il il and tnf-α, which induce migration and proliferation of periosteum mesenchymal stem cells. these cells differentiate into osteoblasts, the major step in the regenerative process. however, during the individual’s lifetime, both the availability and the ability of these cells to differentiate diminish, leading to incomplete or total absence of tissue regeneration at the fracture site. although physiological details are well understood, the molecular aspects of the differentiation process occurring in the osteoblast lineage from adjacent mesenchymal cells remain unclear. to address this issue, autologous mesenchymal stem cells have been utilized, improving the bone tissue regeneration capability and leading to reduction of both total costs and hospitalization period, with a significant decrease in lesion recurrence. these cells gained importance in regenerative medicine, due to their ability to differentiate into chondrocytes, adipocytes and osteoblasts, and facility with which they may be isolated from several organs, among which is the skin. due to its function of protecting from exposure to deleterious agents, such as uv light, physical injuries and pathogens, the skin displays a high cell proliferation rate, which is maintained by the self-renewal and differentiation capabilities of the several stem cell populations present in skin niches. these cells are of particular interest, since they may be easily isolated from the skin, in reasonable amounts, being highly suitable for bone healing and repair. although it is known that osteogenic differentiation in mscs is initiated through activation of canonical pathways such as smad proteins, the possible protein interactions with other pathways which may influence cell differentiation remain elusive. the activation of different downstream signaling cascade pathways, includes hedgehog, wnt, pthr-p and bmps, which, in turn, activate the main transcription factors related to osteogenesis through their respective pathways. smads, for example, may be positively or negatively regulated by phosphorylation of different residues, leading to activation or suppression of the bmp-initiated signal. these kinase pathways, in turn, activate downstream effectors in the cytoplasm and nucleus by phosphorylating a network of substracts. since the study of protein phosphorylation depends mainly on phosphospecific antibodies and the utilization of radioisotopes, identification of novel phosphorylation sites has been a laborious task. however, the development of mass spectrometry techniques by detection of inorganic phosphate neutral loss through cid, originated from phosphoserine, phosphotyrosine and phosphothreonine, has made it possible to describe these novel phosphorylation sites in several proteins. even though the analysis of fragmented peptides using ms tools is an alternative method, due to their negative charge and low abundance, phosphorylated peptides display poor ionization and are subjected to signal suppression, when compared to the regular, non-modified peptides. therefore, it is necessary to enrich for the phosphorylated peptides population present in the sample and to eliminate interfering ions. this may be accomplished by using a metal affinity chromatography, such as imac or tio thus improving the detection levels of modified peptides. this technique, coupled with stable isotope labeling of peptides for quantitative proteomics, may provide information on the proteins which are differentially phosphorylated during bmp2-induced osteodifferentiation. an unexpensive and practical method for quantitative proteomics is the use of stable-isotope dimethyl labeling. primary amine of tryptic peptides and the lysine ϵ-amino group can react with formaldehyde in the presence of cyanoborohydride through reductive amination, giving rise to dimethylated amine as the product. depending on which stable isotope is used, different shifts in molecular mass may be achieved. using both non-modified formaldehyde and cyanoborohydride, the mass shift is of 28 da. using both deuterated formaldehyde and cyanoborohydride, the mass shift is of 32 da, and using 13c plus deuterated formaldehyde and cyanoborohydride, the mass shift is of 36 da. each isotope differs from each other by 4 da per primary amine labeled, with the comparison between different samples being made by ms precursor ion identification on extracted chromatograms. here, we employed mass spectrometry coupled to tio metal affinity chromatography techniques to uncover new players involved in mouse skin mesenchymal cells osteogenic differentiation.  quantitative phosphoproteome and proteome of msmsc cells subjected to rhbmp osteoblastic differentiation msmsc cells cultured in 100 mm dishes were treated with rhbmp for different periods of time, in order to assess protein phosphorylation changes during the first steps of osteoblastic differentiation. previous experiments using the osteoblast differentiation medium showed intense calcification of our murine skin mscs in and 21 days. homogeneity of the skin dermal mscs was probed through a complete characterization of cd markers, namely: cd cd cd cd and cd, utilizing only cell populations displaying greater than 90% purity for the osteogenic differentiation assays. due to the use of three different isotopes to label the samples and five different timepoints, it was necessary to carry out two independent experiments, each of which containing a light, an intermediate and a heavy isotope. we also evaluated protein level changes, through analysis of the total protein fraction in order to compare total protein levels alterations. since the tio flowthrough and wash fractions represent more than 70% of the sample and are highly complex, another fractionation step was performed. hilic separation was used to reduce sample complexity, according to protein hydrophilicity. data analysis the “raw” data acquired from thermo ltq-xl orbitrap® was converted to “.mgf” files and an in-house mascot server was used to search for peptides containing dimethyl and carbamylation as a fixed modification and for phosphorylation in serine, tyrosine and threonine. the thermo proteome discoverer software, version was used to quantify all peptides based on the total area of extracted chromatogram, and the absolute values were normalized using a lowess algorithm. these data were input into the statquant software to evaluate the overall protein ratio by calculating the mean peptide ratio for all peptides corresponding to a given protein. the list for all peptides and phosphopeptides quantified can be accessed in the additional file and a summary of upregulated and downregulated phosphoproteins in each experiment, sorted by period of time indutction with rhbmp is shown in additional file phosphosite localization to assign phosphorylation sites, normalized mascot delta score was used. mascot delta score is the difference between the top two scores for the peptides identified by a given spectrum. dividing this value by the score of the top score peptide, normalized delta score is obtained. in order to have 1% flr for correct phosphosite assignment with 99% certainty, peptides with nmd-score below were discarded. a total of unique phosphosites with 99% certainty that the sites were assigned correctly were identified. these sites were found on different proteins and their distributions were %, % and % for ps, pt and py, respectively, which is comparable to previous works for mammalian cell types. all validated phosphosites with their md-scores are listed in additional file phosphorylation motif database search the analysis carried out to determine which kinase could possibly be involved in phosphsorylation of a given phosphorylated residue from phosphoproteome data was performed using the networkin site. figure  shows a summary of the complete dataset represented by a graph containing kinase motifs occurrencies. network analysis using the ingenuity pathway analysis software in order to evaluate possible intracellular interactors with the phosphopeptides found, a network analysis was performed. the ingenuity pathway analysis software was used to map relationships among proteins, distributed into different cellular compartments. from the total list of proteins found to interact with phosphoproteins, hits containing a transcription factor function were selected for further analysis of dna-binding motifs in osteoblast differentiation-related genes. non-phosphorylated population of peptides were classified according to biological process using the gene ontology blast2go software in order to map and annotate gene ontologies. predicted network of interaction for phosphoproteins found using ingenuity pathway analysis. the list of phosphoproteins found were subjected to ingenuity pathway analysis to investigate problable protein interactions for each cellular compartment. proteins described to be transcription factors were selected to investigate the activation of osteblast related genes by quantitative real-time pcr. consistently upregulated proteins were input in blast2go software for mapping and annotation of gene ontologies. biological processes over-represented were selected and classified according to level. top: low levels; bottom. analysis of the promoter region of osteoblast-related genes for the presence of responsive elements for the bmp2-regulated transcription factors after obtaining the list of transcription factors for the ingenuity® network analysis, a curated database for transcription target genes, tred was used to find target genes and text mining was performed to find which target genes are related with osteoblastic differentiation. we used the jaspar database which contains a curated, non-redundant set of profiles, derived from published collections of experimentally defined transcription factor binding sites for eukaryotes and sorted out the transcription factor which have well defined binding motifs. these motifs were used as a template for a search in the promoter region of the pre-selected genes, using the ensembl cisred database and those which displayed at least one match or multiple matches for the sequences were selected for the qrt-pcr analysis. the consensus sequences of sp c-myc and nfkb were selected among others because they were present in the promoter region in more them 80% of the selected genes for qpcr validation. analysis of differentially expressed genes involved in osteoblastogenesis activated by bmp2-induced transcription factors we used analysis of regulatory networks in order to investigate which transcription factors were activated, and which of them are related with activation of osteoblast-related genes. thirteen genes were selected to evaluate their role in osteoblastic differentiation of msmsc cells, and to confirm the in silico analysis. from the initial list of genes investigated, ten were found to be upregulated at different timepoints. the tgfb cytokine ant its receptor, tgfbr displayed the regulated motifs in their promoter regions. the mrna relative levels of these two genes were evaluated after 10 min, 30 min, 1 h and 2 h of exposure to rhbmp the relative levels of tgfb were upregulated more than two times after 30 min of rhbmp induction, but after reaching this peak, the relative levels decreased to basal levels after 2 h. this pattern was followed by a subsequent increase in the tgfbr mrna relative levels of up to fold at 1 h and more than fold at 2 h. since the synthesis of extracellular matrix compounds, such as collagens, is known to be regulated during osteo differentiation, we selected two members of the collagen family that displayed the selected motifs, namely: collagen and 4a. both ecm components were upregulated, with collagen displaying a punctual increase at 1 h after stimulus and collagen 4a followed a progressively rising pattern. related to collagens and tgfb, the osteogenesis related gene twist presents a downregulation pattern from the basal levels during the beginning of the differentiation and after that a slight increase at 1 h, a decrease to fold at 2 h. expression of smad was accessed, in order to stabilish a possible crosstalk between tgf-beta/activin signaling during bmp2-driven osteogenesis. the mrna relative levels of smad were accessed, presenting a slight increase of fold at 10 min and a major increase of more than fold at 2 h. list of primers used to amplify the cdna in order to investigate changes in mrna upon bmp treatment in qrt-pcr. f: forward; r: reverse. we also evaluated a set of four transcription factors which, in addition to presenting the regulated motifs in their promoter regions, were key elements during the osteoprogenitors differentiation. the relative mrna levels of runx were the first to be upregulated, increasing almost fold after 30 min, with a drastic descent to levels similar to basal levels after 1 h. another important transcription factor, dlx- displayed a progressive increase at 10 min and 30 min reaching a peak at 1 h, followed by a sharp decrease to basal levels at 2 h. the transcription factor osterix displayed a stepwise increase, beginning at 10 min, and reaching up to fold after 2 h of stimulation. similarly, the sox mrna level was upregulated at 30 min and 1 h. discussion in the present study, we used murine skin mesenchymal cells and stable dimethyl isotope labeling to quantify abundant proteins and phosphoproteins using tio metal affinity chromatography, coupled with mass spectrometry, at five different periods of rhbmp induction, namely: and 120 min. from 150 μg of the combined samples, it was possible to identify and quantify distinct phophoproteins and distinct proteins, in all replicates. based on the data acquired, and, also, on references from the literature, we proposed a model for bmp2-mediated osteodifferentiation differentiation of these msmscs cells. previous experiments carried out with these msmscs, subjected to the osteoblast differentiation medium showed intense calcification at and 21 days of treatment, with greater than 80% of the cells being alizarin red-positive”. this experiment could not be carried out solely with bmps-supplemented culture medium, due to its lack of mineral components, which is necessary for mineralization. the data found showed to be compatible with bone development, since bmps act at the very early stages of cells differentiation to the osteoblastic lineage, but, later on in the process, these cells incorporate mineral precursors and originate the calcified bone tissue. the kinases which showed the highest number of phosphorylation motifs in phosphodata were represented, as well as gene activation for each time period studied. we used triplex stable isotope dimethyl labeling to compare five different time periods of rhbmp2-induced osteoblastic differentiation of skin mesenchymal cells, combined into two different experimental groups. this was necessary in order to correctly compare the phosphoprotein ratios with their respective protein levels, since we do not expect a wide protein level variation during the period studied and, also, to avoid aberrations in phosphoprotein variation. similarly, song and colleagues used similar approach through a pseudo-triplex dimethyl labeling in liver tissue samples and hepatocellular carcinoma. each pooled sample was split according to the affinity of peptides for the tio column. the tio2-flowthrough fraction was subjected to hilic and fractionated. since this step decreased the sample complexity, it enhanced the number of peptides which could be identified and quantified, when compared to the entire pool, which was directly injected into the lc/ms. since we started from a relatively low amount of sample, no improvement in the number of detected peptides was obtained using hilic in phospho-enriched samples. analysis of the data also showed that there is a tendency for many phosphopeptides to be upregulated between 30 min and 1 h of rhbmp treatment, correlating with the period of activation for the dlx transcription factors which trigger the expression of runx and osx, both of which are upregulated upon rhbmp administration. in order to compare measurements across lc-ms/ms experiments and to correct for non-biological variation, data normalization is a crucial step prior to any further analysis. the standard normalization assumed in lc/ms experiments is based on dividing all peptide ratio values by log however, notice that this procedure only divides the peptide abundance by a common factor, re-scaling the relative abundance of the peptide. in other words, this “within sample” normalization does not remove the bias in the quantities across experiments. in order to remove the systematic errors introduced in different experiments, we applied the lowess regression, a technique commonly applied to microarray data analysis. one premise to apply lowess normalization is that the differences among the overall intensity of different experiments would be the consequence of non-biological variation, i.e., most peptides will not show a significant change in the abundance between the two compared samples. briefly, in a well performed experiment, the scatter-plot of peptides of one sample versus another would cluster the peptides along a straight line, and the slope would be equal to normalization of these data is equivalent to calculating the best-fit slope using regression techniques and adjusting the intensities so that the calculated slope is however, sometimes, the intensities may be non-linear, therefore, local regression techniques, such as lowess regression, are more suitable. lowess regression is estimated through a locally weighted polynomial regression for a subset of peptides in the neighborhood of each peptide. for more details, please refer to. bmp induces phosphorylation of substrates for different kinases in msmscs kinase prediction analysis using the networkin database, from the phosphorylated peptides found, suggested that, three major kinases could be acting as effectors of phosphorylation upon bmp treatment, namely: casein kinase ii, p mapk and jnk. these kinases are followed, to a lesser extent, by activin receptors and the cdk family of kinases. these data are in agreement with the literature of the field, since bragdon and colleagues showed the involvement of ck in bmp2-induced cells. the release of ck from bmp receptor type i is related with osteblastogenesis, since specific peptides which interfere with this interaction, destabilize the ck2-bmpri complex and enhance osteoblastic differentiation. it is possible that the role of ck in osteogenesis is much more than its release from bmpri, involving many of the substrates found in this work and even other ones which could contribute to the enrollment of these undifferentiated stem cells to osteoblastogenesis. the involvement of p mapk in bmp2-driven osteoblatogenesis is well-known. several studies show activation of p within the first hour of bmp induction, and activation of dlx and osx, essential genes involved in osteblastic differentiation, as well as alkaline phosphatase. we confirmed these data in our model using quantitative real-time pcr experiments, showing an increase in mrna relative expression for osx and dlx it is interesting to note that p may be involved in phosphorylation of several phosphoproteins found in our study, since sites were predicted to be phosphorylated by this kinase. upon bmp treatment, jnk may also be activated, as previous studies described. we found that 9% of all sites could be phosphorylated by this kinase up to 2 h of bmp treatment. interestingly, jnk is transiently activated in mc3t3-e1cells, in a short window, stimulating the expression of osteocalcin. however, at late periods of bmp induction, jnk acts inhibiting the runx function by its phosphorylation at ser in c2c cells. these results show the dual function of jnk in osteoblastogenesis, which is regulated in a time-dependent manner. at early periods of time, jnk may have a role inducing osteogenesis, by phosphorylating intracellular substrates and augmenting the cellular sensibility for bmp. on the other hand, at late periods, jnk would participate by slowing down the intracellular signaling for osteodiffentiation. similar number of phosphorylated sites were found for the cdk group of kinases. these kinases are related with cell cycle progression, and their activation or inhibition is associated with proliferation and quiescence, respectively. at a first glance, the activity of cdk kinases could lead to an impairment of osteoblastic differentiation, due to stimulation of cell proliferation. the role of cdk in osteoblastic differentiation is not well understood yet, however, its inhibitor, the p protein, has been involved in osteoblastic differentiation since p null mice exhibit enhanced osteoblastic differentiation, and overexpression of p protein delays bone formation. it is possible that p could act independently of cdk, activating or repressing genes in the nucleus, with its role controlling osteoblastic differentiation being more complex than simply regulating the cell cycle. further studies will be required to address this issue and how cdk/p regulation participates in osteoblastic differentiation. biological processes overrepresented in bmp2-treated msmscs the proteomic data obtained were analyzed using the gene ontology classification. we observed which gene ontologies could be representative of the upregulated genes. surprisingly, we found a high number of ontologies containing the following terms: multicellular organismal and anatomical structure development, signal transduction signaling, cell differentiation, cell surface receptor linked signaling pathway and phosphorylation at the first hour of bmp treatment, in contrast with the first and 30 min periods of induction, which showed a few gene ontologies with these terms assigned. this can be due to the fact that short periods of time are not sufficient to change the overall amount of protein in the cell, therefore, transcription and translation of new proteins must take place before we can observe changes in protein levels, which are sufficient to affect the gene ontologies classification observed. nevertheless, comparing the second hour of bmp induction with the first one, less gene ontologies could be classified, leading to the conclusion that these proteins involved with signaling are regulated within the first hour bmp induction. bmp2-treated msmscs phosphorylate intracellular messengers which, in turn, activate osteoblastic-related genes bmp induction was shown to modify the post-translational modifications of intracelular proteins, at the timepoints studied. in order to investigate how these phosphorylated proteins activate transcription factors, and whether they are related with the activation of osteoblastic genes, a network analysis of proteins found in the phosphoproteome of bmp2-treated msmscs was carried out. through ingenuity network analysis, we found different transcription factors related with the phospho data. however, not all of the transcription factors found were described to have any participation in osteoblast differentiation, or activation of osteoblastic related genes. using a curated database for transcription target genes, tred, a transcription factors binding motifs occurrence, jaspar, and the literature on the field to search for osteoblastic target genes, one by one, we found three transcription factors from the ingenuity output list, displaying important roles in osteoblastogenesis, namely: sp c-myc e nf-ƙb. tgf-β/bmps are widely recognized for their role in bone formation during mammalian development, exhibiting versatile regulatory functions in the body. in accordance with this finding, we observed increased levels of the mrna for both the tgfb cytokine and for its receptor tgfbr. also, signaling transduction by tgf-β/bmps occurs specifically through both canonical smad-dependent pathways and a non-canonical smad-independent signaling pathway. following tgf-β/bmp induction, both the smad and p mapk pathways converge at the runx gene to control mesenchymal precursor cell differentiation, which has also been found to have increased mrna levels. sox a transcription factor of the sex-determining region y -related high mobility group box family of proteins, is crucial for skeletal development and marks all osteoblastic progenitors, being capable of inducing runx expression. however, the role of sox in osteoblastic differentiation is not completely understood. conditional deletion of sox in the limb bud mesenchyme led to the absence of chondrocytes and osteoblasts. contrastingly, when sox was deleted in the neural crest cells that contribute to the craniofacial skeleton, the cells which normally form chondrocytes expressed osteoblasts markers, suggesting the existence of a the bipotential progenitor. however, sox is not expressed by mature osteoblasts and this is the probable cause of its downregulation after 2 h of the stimulus. col and col4a display functions related with the building of the basal membrane for the newly-formed mature bone tissue. a recent report of comparative transcription of various fetal and adult mesenchymal stem cells sources through quantitative pcr profiling unveiled that collagens, such as collagen and were upregulated during several types of osteogenic differentiation, such as the one reported in this manuscript with the levels of these two extracellular matrix components being increased. supporting these findings, it has been reported that site mutations in collagen leads to high bone mass in osteogenesis imperfecta. since the bhlh transcription factor twist inhibits osteoblast differentiation through binding to a dna-binding domain in runx, the early downregulation of this gene to levels below the basal level at and 30 min could be indicative that the differentiation process was mediated by runx moreover, it has been shown that runx a runt domain-containing transcription factor, is indispensable for osteoblastic differentiation during both endochondral and intramembranous ossification and the function of mature osteoblasts, including the synthesis of bone matrix. homozygous deletion of runx in mice resulted in a complete lack of osteoblasts. our results show a sustained increase in the mrna levels of this transcription factor after 30 min, pointining to the involvement of this gene in the osteogenesis induced by exposure to bmp another essential gene related with osteoblastic differentiation is osx, a transcription factor containing three zinc-fingers. osx was discovered as a bmp-induced gene in c2c cells, with its deletion resulting in complete absence of osteoblasts in mouse embryos, despite the relatively normal expression of runx, which indicates that osx is activated after runx during osteoblastic differentiation. in accordance, we observed that after a significant increase in runx after 30 min of induction, a consistent increase of mrna osx levels is observed up to 2 h after stimulus. finally, we analized the expression levels of dlx- an homeobox gene that plays an essential role in craniofacial, axial, and appendicular skeletal development, and specifically regulates runx expression by binding to the homeodomain-response elements in the runx distal promoter. the increased amounts of dlx- after exposure to bmp indicates that this gene is also present in our differentiation event, generating a reliable axis between dlx-5/runx-2/osx. novel phosphorylated candidates found upon bmp treatment of msmscs from all three independent experiments, we chose proteins which displayed increased phosphorylation upon bmp treatment: a group of proteins related with cytoskeletal rearrangement and ras protein signal transduction. cytoskeletal rearrangement is observed during osteoblastic differentiation through the shift from a fibroblast-like to a spheric phenotype, upon induction with supplemented osteogenic differentiation medium, being antagonized by treatment with cytochalasin d, leading to a reduction of differentiation markers expression. thus, catenin alpha alpha parvin, septin- caldesmon, microtubule associated proteins 1b and nexilin, cytoplasmic dynein light intermediate chain and isoforms of lamin a/c and plectin were found to be upregulated at all time periods studied. together with the previous studies which had described activation of these proteins using odm, we found that these proteins were also activated upon bmp treatment. this may be explained by the fact that a common subset of proteins can be activated by both bmp and components of odm, phosphorylating other proteins related which cytoskeletal rearrangement. another protein related with cytoskeletal rearrangement found in our experiments was rho gtpase-activating protein. the rho family of gtpases plays an important role in osteoblastic differentiation, shown by differentiation to osteogenesis of constitutively rhoa-expressing mesenchymal stem cells. other proteins involving signaling pathways in osteoblastic differentiation were positively phosphorylated, namely: transforming growth factor beta induced transcript and bcl-2-associated transcription factor displayed increasing phosphorylation levels. these proteins are related to the wnt pathway and, specifically, hic- was involved in regulation of intracellular signals by smad and effector proteins of the canonical bmp signaling pathway.  stable isotope dimethyl labeling of peptides may be used to quantify small amounts of proteins phosphorylated in cell extracts. during bmp2-induced differentiation in skin derived mesenchymal stem cells, it was possible to acess different proteins, which many of them were found to be phosphorylated in different timepoints, giving new cues about the events that occur in the short term of osteoblastic differentiation. 
gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after bmp induction. we assessed differences in the phosphorylation status of different cellular proteins upon bmp- osteogenic induction of isolated murine skin mesenchymal stem cells using triplex stable isotope dimethyl labeling coupled with lc/ms. cytokines, such as il- il- and tnfα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins, mainly bmp- although it is known that bmp- binds to actri/bmpr and activates the smad 1/5/ downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to bmp2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells.
 bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. cytokines, such as il- il- and tnfα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins, mainly bmp- although it is known that bmp- binds to actri/bmpr and activates the smad 1/5/ downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. we assessed differences in the phosphorylation status of different cellular proteins upon bmp- osteogenic induction of isolated murine skin mesenchymal stem cells using triplex stable isotope dimethyl labeling coupled with lc/ms.  from 150 μg of starting material, proteins were identified and quantified at five different time points, of which are differentially phosphorylated. kinase motif analysis showed that several substrates display phosphorylation sites for casein kinase, p cdk and jnk. gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after bmp induction. moreover, proteins involved in cytoskeleton rearrangement, wnt and ras pathways were found to be differentially phosphorylated during all timepoints studied.  taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to bmp2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells. 