Partial knockdown of Fer cooperated with suboptimal expression of NS- and LS- but not WT Shp2 to induce developmental defects.

<p>A. Embryos were injected with control MO, Fer e9i9 MO and NS, LS or WT Shp2 mRNA as indicated, and imaged at 4 dpf. B. Alcian blue staining showing craniofacial defects scored on severity (green: wild type, yellow: mild phenotype, orange: moderate phenotype, red: severe phenotype). C. Embryos were scored based on craniofacial defect upon alcian blue staining. The number of embryos per condition is indicated. D. The angle of the ceratohyal was quantified as a measure of craniofacial defects (* p<0.005, Student's t-test).</p

Fer knockdown induced craniofacial defects in zebrafish embryos.

<p>A. Embryos were injected at the 1-cell stage with 1 ng Fer i5e6 MO, 2.5 ng e9i9 MO or 2.5 ng Nacre MO as a negative control. Additionally, embryos were injected with 150 ng WT Shp2 RNA, 150 ng NS Shp2 RNA or 50 ng LS Shp2 RNA. B. Embryos were injected at the 1-cell stage with 2.5 ng Nacre control MO, 1.0 ng Fer i5e6 MO, 2.5 ng Fer e9i9 MO or in combination with 2.5 ng P53 MO. C. Craniofacial structures were imaged using <i>Tg(-4.9sox10:EGFP)^ba2</i> embryos expressing eGFP in neural crest cells that also form the cartilage. Embryos were injected with Fer MO e9i9 at the 1 cell stage and imaged at 4dpf. Ceratohyal is indicated with a red asterisk and Meckel's cartilage with a white asterisk. Both moderate and severe phenotypes are depicted together with non-injected controls (NIC). D. Embryos were injected at the 1-cell stage with suboptimal concentrations of MO (0.5 ng i5e6 MO; <i>n</i> = 110 and 1.0 ng e9i9 MO; <i>n</i> = 99). High levels of both MO's (1.0 ng i5e6 MO; <i>n</i> = 122 and 2.5 e9i9 MO; <i>n</i> = 135) or low levels of both MO's were co-injected (<i>n</i> = 115). Embryos were imaged at 3 dpf and 4 dpf and grouped by having a WT appearance (green), a craniofacial defect alone (yellow) a heart defect alone (orange) or both (red) at 4 dpf. Relative levels of phenotypes are depicted. E. Embryos were injected at the 1-cell stage with normal dose of MO (1.0 ng i5e6 MO; <i>n</i> = 61 and 2.5 e9i9 MO; <i>n</i> = 53), low doses of MO (0.5 ng i5e6 MO; <i>n</i> = 57 and 1.0 ng e9i9 MO; <i>n</i> = 50) or a co-injected with low doses of MO (<i>n</i> = 70). Morphology at 4 dpf is depicted. Embryos were fixed and stained with Alcian blue at 4 dpf and imaged laterally and ventrally. For quantification, the angle of the ceratohyal (F) and the ratio of the distance from the back of the head to Meckel's cartilage and the width of the head was determined (G) (* indicates significance, Student's t-test p<0.005).</p

Comparative mass-spectrometry of pTyr immunoprecipitated zebrafish lysates.

<p>Zebrafish embryos were injected at the 1-cell stage with synthetic mRNA constructs encoding WT Shp2, NS (D61G) Shp2 or LS (A462T) Shp2 and co-injected with mRNA encoding eGFP. Lysates were subjected to mass spectrometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106682#s2" target="_blank">Materials and Methods</a>. Normalized ratios (Log2 scale) based on total levels of non-phosphorylated peptides are given.</p><p>*Protein name based on BLAST sequence of peptide. Accession numbers from BLAST hits are used for non-annotated peptides.</p><p>**pRS score <75, phosphorylation site could not be determined but the most commonly identified site from Phsophosite.org is used. a-n: indicators for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106682#pone-0106682-g001" target="_blank">Figure 1C</a>.</p><p>Comparative mass-spectrometry of pTyr immunoprecipitated zebrafish lysates.</p

Fer knockdown resulted in C&E defects but not in changes in cell fate.

<p>A. <i>Krox20/myoD in situ</i> hybridization as a method to quantify C&E defects. <i>Krox20</i> (red) staining for rhombomeres 3 and 5 was used to measure the width, which correlates with convergence cell movements in the embryo. <i>MyoD</i> (light blue) staining for the somites was used to measure the length, <i>i.e.</i> extension of the embryo. The ratio of <i>Krox20/myoD</i> correlates directly with convergence & extension cell movements during gastrulation. B. Flatmounts of <i>krox20/myoD</i> stained NIC (<i>n</i> = 70), control MO (<i>n</i> = 44) and Fer MO (<i>n</i> = 40) embryos. C. The ratio of the width of a krox20-positive rhombomere and the length of 8 somites was determined (* indicates significance, Student's t-test p<0.005). D–X. Embryos were injected at the 1-cell stage with control MO or Fer e9i9 MO and subjected to ISH for various markers of cell fate determination. Note that the staining of the <i>gsc, pax2, six3</i> and <i>cyc</i> probes show broader and shorter expression in Fer knockdown embryos than in controls.</p

Fer expression in zebrafish embryos and Fer MO induced splicing defects.

<p>A. Alignment of zebrafish Fer kinase sequence encompassing the autophosphorylation site (black) that was identified by phosphoproteomics with the mammals <i>M. musculus</i>, <i>R. norvegicus</i>, <i>H. sapiens</i>, the avian <i>G. gallus</i>, the amphibian <i>Xenopus tropicalis</i>, and the fish <i>Tetraodon nigrovirides, Takifugu rupripes</i> and <i>Oryzias latipes</i>. B. <i>fer</i> expression during the first 24 hpf and at 3 dpf and 4 dpf was observed using <i>in situ</i> hybridization with an antisense <i>fer</i> probe. C. ISH with negative control sense <i>fer</i> probe. D. RT-PCR showing altered splicing of <i>fer</i> in MO injected embryos. Da. Model of altered splicing by Fer MO i5e6. Exons 5, 6 and 7 are indicated in red, blue and green, respectively. Primers used for RT-PCR are indicated as arrows. MO is indicated in red. Due to splice blocking, intron 5 is not spliced out of the processed mRNA. Db. RT-PCR showing <i>gapdh</i> control in Nacre (control) MO and Fer MO injected embryos. RT-PCR showing <i>Fer</i> product in Nacre control MO and Fer i5e6 MO injected embryos, and genomic DNA as a positive control. Dc. Sanger sequencing showing the inclusion of intron 5 and the normal splicing of exon 6 in the RT-PCR product. Dd. Model of altered splicing by Fer MO e9i9. Exons 8, 9 and 10 are indicated in red, blue and green, respectively. Primers used for RT-PCR are indicated as arrows. MO is indicated in red. Due to defective splicing, exon 9 is spliced out of the processed mRNA. De. RT-PCR showing <i>gapdh</i> control in Nacre (control) MO and Fer e9i9 MO injected embryos. RT-PCR showing <i>Fer</i> product in Nacre control MO and Fer e9i9 MO injected embryos with a decrease in size of the product in e9i9 MO injected embryos. Df. Sanger sequencing showing the exclusion of exon 9.</p

Comparative pTyr mass spectrometry on 1 day old zebrafish embryos expressing wild type, NS (D61G) or LS (A462T) Shp2.

<p>A. Work flow depicting the mass spectrometry approach. Approximately 2000 zebrafish embryos per condition were injected at the 1-cell stage and sorted for GFP expression. Embryos were lysed, trypsinised and labeled using the dimethyl labeling method. WT, NS and LS samples were combined and immunoprecipitated using pTyr specific antibodies. Immunoprecipitate was subjected to MS and peptides were identified and quantified based on MS2 and MS1 spectra, respectively. B. 1 dpf zebrafish embryos expressing WT, D61G and A462T Shp2. Body axis length, craniofacial defects and heart edema in D61G and A462T Shp2 expressing zebrafish are indicated with arrowheads. C. Normalized plot of quantified phosphopeptides Log2 ratios. Peptide ratios with Log2 ratios >−1 and <1 are indicated in black. Peptides with more that 1× Log2 difference are annotated with a-n (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106682#pone-0106682-t001" target="_blank">Table 1</a> for reference). Peptides changed with Log2 ratios <−1 in either NS or LS are indicated in green and Log2 ratios >1 in either NS or LS are indicated in red. See text for further details.</p

Opposite Electron-Transfer Dissociation and Higher-Energy Collisional Dissociation Fragmentation Characteristics of Proteolytic K/R(X)_<i>n</i> and (X)<i>_n</i>K/R Peptides Provide Benefits for Peptide Sequencing in Proteomics and Phosphoproteomics

A key step in shotgun proteomics is the digestion of proteins into peptides amenable for mass spectrometry. Tryptic peptides can be readily sequenced and identified by collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) because the fragmentation rules are well-understood. Here, we investigate LysargiNase, a perfect trypsin mirror protease, because it cleaves equally specific at arginine and lysine residues, albeit at the N-terminal end. LysargiNase peptides are therefore practically tryptic-like in length and sequence except that following ESI, the two protons are now both positioned at the N-terminus. Here, we compare side-by-side the chromatographic separation properties, gas-phase fragmentation characteristics, and (phospho)­proteome sequence coverage of tryptic (i.e., (X)_<i>n</i>K/R) and LysargiNase (i.e., K/R­(X)_<i>n</i>) peptides using primarily electron-transfer dissociation (ETD) and, for comparison, HCD. We find that tryptic and LysargiNase peptides fragment nearly as mirror images. For LysargiNase predominantly N-terminal peptide ions (c-ions (ETD) and b-ions (HCD)) are formed, whereas for trypsin, C-terminal fragment ions dominate (z-ions (ETD) and y-ions (HCD)) in a homologous mixture of complementary ions. Especially during ETD, LysargiNase peptides fragment into low-complexity but information-rich sequence ladders. Trypsin and LysargiNase chart distinct parts of the proteome, and therefore, the combined use of these enzymes will benefit a more in-depth and reliable analysis of (phospho)­proteomes

Opposite Electron-Transfer Dissociation and Higher-Energy Collisional Dissociation Fragmentation Characteristics of Proteolytic K/R(X)_<i>n</i> and (X)<i>_n</i>K/R Peptides Provide Benefits for Peptide Sequencing in Proteomics and Phosphoproteomics

A key step in shotgun proteomics is the digestion of proteins into peptides amenable for mass spectrometry. Tryptic peptides can be readily sequenced and identified by collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) because the fragmentation rules are well-understood. Here, we investigate LysargiNase, a perfect trypsin mirror protease, because it cleaves equally specific at arginine and lysine residues, albeit at the N-terminal end. LysargiNase peptides are therefore practically tryptic-like in length and sequence except that following ESI, the two protons are now both positioned at the N-terminus. Here, we compare side-by-side the chromatographic separation properties, gas-phase fragmentation characteristics, and (phospho)­proteome sequence coverage of tryptic (i.e., (X)_<i>n</i>K/R) and LysargiNase (i.e., K/R­(X)_<i>n</i>) peptides using primarily electron-transfer dissociation (ETD) and, for comparison, HCD. We find that tryptic and LysargiNase peptides fragment nearly as mirror images. For LysargiNase predominantly N-terminal peptide ions (c-ions (ETD) and b-ions (HCD)) are formed, whereas for trypsin, C-terminal fragment ions dominate (z-ions (ETD) and y-ions (HCD)) in a homologous mixture of complementary ions. Especially during ETD, LysargiNase peptides fragment into low-complexity but information-rich sequence ladders. Trypsin and LysargiNase chart distinct parts of the proteome, and therefore, the combined use of these enzymes will benefit a more in-depth and reliable analysis of (phospho)­proteomes

Evaluating the Promiscuous Nature of Tyrosine Kinase Inhibitors Assessed in A431 Epidermoid Carcinoma Cells by Both Chemical- and Phosphoproteomics

Deregulation of protein tyrosine kinase signaling has been linked to many diseases, most notably cancer. As a consequence, small molecule inhibitors of protein tyrosine kinases may provide powerful strategies for treatment. Following the successful introduction of imatinib in the treatment of chronic myelogenous leukemia, such drugs are also now evaluated for other types of cancer. However, many developed kinase inhibitors are not very target-specific and therefore may induce side effects. The importance of such side effects is certainly cell-proteome dependent. Understanding the all-inclusive action of a tyrosine kinase inhibitor on each individual cell-type entails the identification of potential targets, combined with monitoring the downstream effects revealing the signaling networks involved. Here, we explored a multilevel quantitative mass spectrometry–based proteomic strategy to identify the direct targets and downstream signaling effect of four tyrosine kinase inhibitors (imatinib, dasatinib, bosutinib, and nilotinib) in epidermoid carcinoma cells, as a model system for skin-cancer. More than 25 tyrosine kinases showed affinity to the drugs, with imatinib and nilotinib displaying a high specificity, especially when compared to dasatinib and bosutinib. Consequently, the latter two drugs showed a larger effect on downstream phosphotyrosine signaling. Many of the proteins affected are key regulators in cell adhesion and invasion. Our data represents a multiplexed view on the promiscuous action of certain tyrosine kinase inhibitors that needs to be taking into consideration prior to the application of these drugs in the treatment of different forms of cancer

Осаждение пленок TiN и TiO₂ в обращенном цилиндрическом магнетроне методом реактивного распыления

Рассмотрена возможность контроля условий получения стехиометрических пленок TiN и TiO₂ по спектральным характеристикам плазмы магнетронного разряда и по изменению разрядного напряжения