Additional file 5: Figure S2. of Tribbles ortholog NIPI-3 and bZIP transcription factor CEBP-1 regulate a Caenorhabditis elegans intestinal immune surveillance pathway

Genes differentially expressed in nipi-3(fr4) are enriched for translational inhibitor- and pathogen-response genes. a. Overlaps between genes differentially expressed in nipi-3(fr4) versus wild type animals fed the indicated food. Numbers provided for major overlap classes. b. Overlap between genes differentially expressed in nipi-3(fr4) versus wild type animals fed control OP50 E. coli and genes induced/repressed by ToxA or hygromycin versus control bacteria in wild type animals [5]. P < 1 × 10–95 (hypergeometric test). c. Overlap between genes differentially expressed in nipi-3(fr4) versus wild type animals fed P. aeruginosa or ToxA and genes induced/repressed by P. aeruginosa or ToxA versus control bacteria in wild type animals P < 5 × 10–6 (hypergeometric test). Data collected from microarray (b) or NanoString (a, c) analyses. Primary data for panels a and c are provided in Additional file 15. (PDF 403 kb

It Makes Me Spit: The Public and Newspaper Reaction to the UK Governmentr's Threat to Suppress the Daily Mirror

cebp-1 acts in the intestine and affects immune gene expression. a. Lifespans of MGH167 (gut RNAi) animals grown on equal mixtures of cebp-1 RNAi and L4440 vector control; nipi-3 RNAi and L4440 vector control; cebp-1 and nipi-3 RNAi; or L4440 vector control alone to the L4 stage. Animals were then transferred to E. coli expressing ToxA. Note that the mixed nipi-3 RNAi showed less ToxA susceptibility than undiluted nipi-3 RNAi (Fig. S1b). P = 0.95 (log-rank test) and 0.4 (Wilcoxon test) for nipi-3, cebp-1 versus cebp-1 RNAi; P = 0.089 (log-rank test) and 0.0004 (Wilcoxon test) for nipi-3 versus cebp-1 RNA. Number of animals scored for each condition was > 65 (426 total). This is a representative experiment of two independent experiments. b. qRT-PCR comparison of the indicated strains exposed to E. coli expressing ToxA for 24 hours. Results shown are an average of two biological replicates and are normalized to the corresponding wild type ToxA value. Error bars represent SEM. nipi-3 refers to nipi-3(fr4). Primary data for panel b are provided in Additional file 15. (PDF 206 kb

Accurate Multiplexed Proteomics at the MS2 Level Using the Complement Reporter Ion Cluster

Isobaric labeling strategies, such as isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tags (TMT), have promised to dramatically increase the power of quantitative proteomics. However, when applied to complex mixtures, both the accuracy and precision are undermined by interfering peptide ions that coisolate and cofragment with the target peptide. Additional gas-phase isolation steps, such as proton-transfer ion–ion reactions (PTR) or higher-order MS3 scans, can almost completely eliminate this problem. Unfortunately, these methods come at the expense of decreased acquisition speed and sensitivity. Here we present a method that allows accurate quantification of TMT-labeled peptides at the MS2 level without additional ion purification. Quantification is based on the fragment ion cluster that carries most of the TMT mass balance. In contrast to the use of low <i>m</i>/<i>z</i> reporter ions, the localization of these complement TMT (TMT^C) ions in the spectrum is precursor-specific; coeluting peptides do not generally affect the measurement of the TMT^C ion cluster of interest. Unlike the PTR or MS3 strategies, this method can be implemented on a wide range of high-resolution mass spectrometers like the quadrupole Orbitrap instruments (QExactive). A current limitation of the method is that the efficiency of TMT^C ion formation is affected by both peptide sequence and peptide ion charge state; we discuss potential routes to overcome this problem. Finally, we show that the complement reporter ion approach allows parallelization of multiplexed quantification and therefore holds the potential to multiply the number of distinct peptides that can be quantified in a given time frame

Additional file 9: Figure S4. of Tribbles ortholog NIPI-3 and bZIP transcription factor CEBP-1 regulate a Caenorhabditis elegans intestinal immune surveillance pathway

Comparing RNA and protein changes in animals exposed to ToxA or control bacteria. Changes of protein and RNA abundances in wild type N2 animals following a 24 hour exposure to E. coli expressing ToxA as compared to animals fed a control BL21 E. coli (top) or in nipi-3(fr4) animals fed control bacteria as compared to similarly treated wild type animals (bottom). Only values with significant protein and/or RNA changes are included. Results shown are an average of two (protein) or three (RNA) biological replicates. Primary data are provided in Additional file 8: Table S5. (PDF 423 kb

Generation of Multiple Reporter Ions from a Single Isobaric Reagent Increases Multiplexing Capacity for Quantitative Proteomics

Isobaric labeling strategies for mass spectrometry-based proteomics enable multiplexed simultaneous quantification of samples and therefore substantially increase the sample throughput in proteomics. However, despite these benefits, current limits to multiplexing capacity are prohibitive for large sample sizes and impose limitations on experimental design. Here, we introduce a novel mechanism for increasing the multiplexing density of isobaric reagents. We present Combinatorial Isobaric Mass Tags (CMTs), an isobaric labeling architecture with the unique ability to generate multiple series of reporter ions simultaneously. We demonstrate that utilization of multiple reporter ion series improves multiplexing capacity of CMT with respect to a commercially available isobaric labeling reagent with preserved quantitative accuracy and depth of coverage in complex mixtures. We provide a blueprint for the realization of 16-plex reagents with 1 Da spacing between reporter ions and up to 28-plex at 6 mDa spacing using only 5 heavy isotopes per reagent. We anticipate that this improvement in multiplexing capacity will further advance the application of quantitative proteomics, particularly in high-throughput screening assays

Multiplexed <i>in Vivo</i> His-Tagging of Enzyme Pathways for <i>in Vitro</i> Single-Pot Multienzyme Catalysis

Protein pathways are dynamic and highly coordinated spatially and temporally, capable of performing a diverse range of complex chemistries and enzymatic reactions with precision and at high efficiency. Biotechnology aims to harvest these natural systems to construct more advanced <i>in vitro</i> reactions, capable of new chemistries and operating at high yield. Here, we present an efficient Multiplex Automated Genome Engineering (MAGE) strategy to simultaneously modify and co-purify large protein complexes and pathways from the model organism <i>Escherichia coli</i> to reconstitute functional synthetic proteomes <i>in vitro</i>. By application of over 110 MAGE cycles, we successfully inserted hexa-histidine sequences into 38 essential genes <i>in vivo</i> that encode for the entire translation machinery. Streamlined co-purification and reconstitution of the translation protein complex enabled protein synthesis <i>in vitro</i>. Our approach can be applied to a growing area of applications in <i>in vitro</i> one-pot multienzyme catalysis (MEC) to manipulate or enhance <i>in vitro</i> pathways such as natural product or carbohydrate biosynthesis

Increasing the Multiplexing Capacity of TMTs Using Reporter Ion Isotopologues with Isobaric Masses

Quantitative mass spectrometry methods offer near-comprehensive proteome coverage; however, these methods still suffer with regards to sample throughput. Multiplex quantitation via isobaric chemical tags (e.g., TMT and iTRAQ) provides an avenue for mass spectrometry-based proteome quantitation experiments to move away from simple binary comparisons and toward greater parallelization. Herein, we demonstrate a straightforward method for immediately expanding the throughput of the TMT isobaric reagents from 6-plex to 8-plex. This method is based upon our ability to resolve the isotopic shift that results from substituting a ¹⁵N for a ¹³C. In an accommodation to the preferred fragmentation pathways of ETD, the TMT-127 and -129 reagents were recently modified such that a ¹³C was exchanged for a ¹⁵N. As a result of this substitution, the new TMT reporter ions are 6.32 mDa lighter. Even though the mass difference between these reporter ion isotopologues is incredibly small, modern high-resolution and mass accuracy analyzers can resolve these ions. On the basis of our ability to resolve and accurately measure the relative intensity of these isobaric reporter ions, we demonstrate that we are able to quantify across eight samples simultaneously by combining the ¹³C- and ¹⁵N-containing reporter ions. Considering the structure of the TMT reporter ion, we believe this work serves as a blueprint for expanding the multiplexing capacity of the TMT reagents to at least 10-plex and possibly up to 18-plex

Large-Scale Proteomic Characterization of Melanoma Expressed Proteins Reveals Nestin and Vimentin as Biomarkers That Can Potentially Distinguish Melanoma Subtypes

Melanoma is an aggressive type of skin cancer, which accounts for only 4% of skin cancer cases but causes around 75% of skin cancer deaths. Currently, there is a limited set of protein biomarkers that can distinguish melanoma subtypes and provide an accurate prognosis of melanoma. Thus, we have selected and profiled the proteomes of five different melanoma cell lines from different stages of progression in comparison with a normal melanocytes using tandem mass spectrometry. We also profiled the proteome of a solid metastatic melanoma tumor. This resulted in the identification of 4758 unique proteins, among which ∼200–300 differentially expressed proteins from each set were found by quantitative proteomics. Correlating protein expression with aggressiveness of each melanoma cell line and literature mining resulted in the final selection of six proteins: vimentin, nestin, fibronectin, annexin A1, dipeptidyl peptidase IV, and histone H2A1B. Validation of nestin and vimentin using 40 melanoma samples revealed pattern of protein expression can help predict melanoma aggressiveness in different subgroups of melanoma. These results, together with the combined list of 4758 expressed proteins, provide a valuable resource for selecting melanoma biomarkers in the future for the clinical and research community

Supplementary table 2.xlsx

<p><b>Lipidomic data </b> <b></b></p><p>Lipidomic maps were generated for mouse KGC pancreatic tumor organoid lines expressing doxycycline-inducible constitutively-active<b> </b>mutant GNAS (GNAS^R201C). The organoids were analyzed in the presence of doxycycline; Dox(+) or absence of doxycycline; Dox(-). LC-MS experiments for non-polar metabolites were performed using a KCG organoid line in triplicate for the +Dox and -Dox conditions and denoted as Dox(+)-1; Dox(+)-2; Dox(+)-3; Dox(-)-1; Dox(-)-2; Dox(-)-3. LC-MS/MS-based lipidomic experiments and data analyses were performed as described in the reference¹. The data were normalized to the total number of cells in the organoids from replicate wells. Metabolites were quantified by integrating the area under the curve. Lipidation (lipid species detected); Class (class of lipids. see lipid keys in the document)</p><p>Reference: 1) Smulan, L. J.<i>et al.</i>Cholesterol-Independent SREBP-1 Maturation Is Linked to ARF1 Inactivation. <i>Cell reports</i><b>16</b>, 9-18, doi:10.1016/j.celrep.2016.05.086 (2016).</p

Supplementary table 3

<p><b>Polar metabolite data</b></p><p>Polar metabolite profiling data were generated for a mouse KGC pancreatic tumor line expressing doxycycline-inducible constitutively-active<b> </b>mutant GNAS (GNAS^R201C) growing in 2D culture in the presence of doxycycline; Dox(+) or absence of doxycycline; Dox(-). The cells were harvested as 5 biological replicates and flash-frozen. The polar metabolites were extracted with 40:40:20 acetonitrile/methanol/water and replicates are denoted as Dox(+)-1, Dox(+)-2, Dox(+)-3, Dox(+)-4, Dox(+)-5 and Dox(-)-1, Dox(-)-2, Dox(-)-3, Dox(-)-4, Dox(-)-5. Polar metabolites were analyzed by QQQ-LC/MS/MSas described in the reference^1.The ion counts were normalized to the total cell number. Metabolites were quantified by integrating the area under the curve, and then normalized to internal standard values. </p><p> </p><p>1) Louie, S. M.<i>et al.</i>GSTP1 Is a Driver of Triple-Negative Breast Cancer Cell Metabolism and Pathogenicity. <i>Cell Chem Biol</i><b>23</b>, 567-578, doi:10.1016/j.chembiol.2016.03.017 (2016). </p