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Elucidation of activation mechanisms governing protein fusions is essential for therapeutic development. MLL undergoes rearrangement with numerous partners, including a recurrent translocation fusing the epigenetic regulator to a cytoplasmic RAS effector, AF6/afadin. We show here that AF6 employs a non-canonical, evolutionarily conserved α-helix to bind RAS, unique to AF6 and the classical RASSF effectors. Further, all patients with MLL-AF6 translocations express fusion proteins missing only this helix from AF6, resulting in exposure of hydrophobic residues that induce dimerization. We provide evidence that oligomerization is the dominant mechanism driving oncogenesis from rare MLL translocation partners and employ our mechanistic understanding of MLL-AF6 to examine how dimers induce leukemia. Proteomic data resolve association of dimerized MLL with gene expression modulators, and inhibiting dimerization disrupts formation of these complexes while completely abrogating leukemogenesis in mice. Oncogenic gene translocations are thus selected under pressure from protein structure/function, underscoring the complex nature of chromosomal rearrangements. [ABSTRACT FROM AUTHOR]

As a result of recent improvements in mass spectrometry (MS), there is increased interest in data-independent acquisition (DIA) strategies in which all peptides are systematically fragmented using wide mass-isolation windows ('multiplex fragmentation'). DIA-Umpire (http://diaumpire.sourceforge.net/), a comprehensive computational workflow and open-source software for DIA data, detects precursor and fragment chromatographic features and assembles them into pseudo-tandem MS spectra. These spectra can be identified with conventional database-searching and protein-inference tools, allowing sensitive, untargeted analysis of DIA data without the need for a spectral library. Quantification is done with both precursor- and fragment-ion intensities. Furthermore, DIA-Umpire enables targeted extraction of quantitative information based on peptides initially identified in only a subset of the samples, resulting in more consistent quantification across multiple samples. We demonstrated the performance of the method with control samples of varying complexity and publicly available glycoproteomics and affinity purification-MS data. [ABSTRACT FROM AUTHOR]

Abstract: Affinity purification coupled to mass spectrometry (AP-MS) is an effective means of identifying protein–protein interactions to better understand biological functions. However, issues associated with sample preparation still limit the success of AP-MS for specific classes of proteins, including those associated with chromatin that exhibit overall poor solubility in the protocols normally used for AP-MS analysis. Here, we wanted to provide a generally applicable method to simultaneously identify interactors for the chromatin-bound and the soluble fractions of a given bait protein. Using four FLAG-tagged canonical histone proteins (H2A, H2B, H3.1 and H4) we demonstrate that the chromatin solubility issue can be robustly alleviated by fragmenting DNA prior to AP-MS using a combination of sonication and nuclease treatment. We show that – in comparison to a commonly used AP-MS method – our optimized protocol greatly improves the recovery of chromatin-associated interactors for core histones. Critically, this is achieved while preserving the interaction partners associated with the soluble portion of the histones. Detailed protocols amenable to the study of both histone and non-histone baits are presented here. Biological significance: This manuscript describes workflow improvements to enable the recovery of chromatin-bound interactors by affinity purification coupled to mass spectrometry (AP-MS). This is significant, as most of the high-throughput studies to date can only monitor protein–protein interactions for soluble (not bound to chromatin) components. By consequence, we still poorly understand how protein complexes form on chromatin, which greatly hampers our understanding of gene expression. Using core histones as test cases, we show here a simple and universally applicable workflow that permits the identification of chromatin-bound protein–protein interactions. As exemplified in our manuscript, this revised protocol should result in a much deeper understanding of chromatin biology. This article is part of a Special Issue: Can Proteomics Fill the Gap Between Genomics and Phenotypes? [Copyright &y& Elsevier]

The Empirical Statistical Model (ESM) for decoy library searching fused the expected amino acid sequence of 18 non-human protein standards to a human decoy library. The ESM assumed a priori the standards were pure such that only the 18 nominal proteins were true positive, all other proteins were false positive, there was no overlap in the peptides of non-human proteins versus human proteins, and that the score distribution of individual peptides would resolve true positive from false positive results or noise. The results of random and independent sampling by LC-ESI-MS/MS indicated that the fundamental assumptions of the ESM were not in good agreement with the actual purity of the commercial test standards and so the method showed a 99.7% false negative rate. The ESM for decoy library searching apparently showed poor agreement with SDS-PAGE using silver staining, goodness of fit of MS/MS spectra by X!TANDEM, FDR correction by Benjamini and Hochberg, or comparison to the observation frequency of null random MS/MS spectra, that all confirmed the standards contain hundreds of proteins with a low FDR of primary structural identification. The protein observation frequency increased with abundance and the log 10 precursor intensity distributions were Gaussian and nearly ideal for relative quantification. Image 1 • The ESM that is the basis of the entirely novel decoy library method for FDR showed a 99.7% false negative rate. • Protein standards were analyzed by SDS-PAGE, LC-ESI-MS/MS and compared to random MS/MS spectra controls. • SDS-PAGE, LC-ESI-MS/MS and random MS/MS spectra controls agree the protein standards contain hundreds of proteins. • X!TANDEM protein p-value and FDR q-value from R statistical analysis controls error rates. • Linear quadrupole ion trap was sufficient for identification and quantification of proteins with low type I error rate. [ABSTRACT FROM AUTHOR]

Background: It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma by using liquid chromatography and tandem mass spectrometry to identify, quantify and compare the peptides cleaved ex vivo from different clinical populations. The endogenous tryptic peptides of ovarian cancer plasma were compared to breast cancer and female cancer normal controls, other diseases with their matched or normal controls, plus ice cold plasma to control for pre-analytical variation. Methods: The endogenous tryptic peptides or tryptic phospho peptides (i.e. without exogenous digestion) were analyzed from 200 μl of EDTA plasma. The plasma peptides were extracted by a step gradient of organic/water with differential centrifugation, dried, and collected over C18 for analytical HPLC nano electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) with a linear quadrupole ion trap. The endogenous peptides of ovarian cancer were compared to multiple disease and normal samples from different institutions alongside ice cold controls. Peptides were randomly and independently sampled by LC–ESI–MS/MS. Precursor ions from peptides > E4 counts were identified by the SEQUEST and X!TANDEM algorithms, filtered in SQL Server, before testing of frequency counts by Chi Square (χ2), for analysis with the STRING algorithm, and comparison of precursor intensity by ANOVA in the R statistical system with the Tukey-Kramer Honestly Significant Difference (HSD) test. Results: Peptides and/or phosphopeptides of common plasma proteins such as HPR, HP, HPX, and SERPINA1 showed increased observation frequency and/or precursor intensity in ovarian cancer. Many cellular proteins showed large changes in frequency by Chi Square (χ2 > 60, p < 0.0001) in the ovarian cancer samples such as ZNF91, ZNF254, F13A1, LOC102723511, ZNF253, QSER1, P4HA1, GPC6, LMNB2, PYGB, NBR1, CCNI2, LOC101930455, TRPM5, IGSF1, ITGB1, CHD6, SIRT1, NEFM, SKOR2, SUPT20HL1, PLCE1, CCDC148, CPSF3, MORN3, NMI, XTP11, LOC101927572, SMC5, SEMA6B, LOXL3, SEZ6L2, and DHCR24. The protein gene symbols with large Chi Square values were significantly enriched in proteins that showed a complex set of previously established functional and structural relationships by STRING analysis. Analysis of the frequently observed proteins by ANOVA confirmed increases in mean precursor intensity in ZFN91, TRPM5, SIRT1, CHD6, RIMS1, LOC101930455 (XP_005275896), CCDC37 and GIMAP4 between ovarian cancer versus normal female and other diseases or controls by the Tukey–Kramer HSD test. Conclusion: Here we show that separation of endogenous peptides with a step gradient of organic/water and differential centrifugation followed by random and independent sampling by LC–ESI–MS/MS with analysis of peptide frequency and intensity by SQL Server and R revealed significant difference in the ex vivo cleavage of peptides between ovarian cancer and other clinical treatments. There was striking agreement between the proteins discovered from cancer plasma versus previous biomarkers discovered in tumors by genetic or biochemical methods. The results indicate that variation in plasma proteins from ovarian cancer may be directly discovered by LC–ESI–MS/MS that will be a powerful tool for clinical research. [ABSTRACT FROM AUTHOR]

Abstract: The simplest model—that authentic tandem mass spectrometry (MS/MS) spectra are no different from noise, random spectra, or false-positive results—may be directly examined by chi-square comparison of the peptide-to-protein distribution. The peptide-to-protein distribution of a set of 4151 redundant blood proteins identified by X!TANDEM indicated that there is a low probability that the authentic data were the same as noise, random spectra, or false-positive correlations (P <0.0001). In contrast, a competition for significance failed to distinguish approximately 90% of authentic blood proteins from those of noise, random spectra, or false-positive results (P <0.01) and apparently incurred a large type II error (false negative). The chi-square test of peptide-to-protein frequency distributions was found to be an efficient means to distinguish authentic data from false-positive results. Frequency-based statistics unambiguously demonstrated that proteins can be identified by liquid chromatography–electrospray ionization-MS/MS from human blood with acceptable confidence. Thus, the chi-square fit of the peptide-to-protein distribution could distinguish authentic data from random or false-positive data, but the score distribution method could not separate real results from false results. [Copyright &y& Elsevier]

Abstract: Proteomics uses tandem mass spectrometers and correlation algorithms to match peptides and their fragment spectra to amino acid sequences. The replication of multiple liquid chromatography experiments with electrospray ionization of peptides and tandem mass spectrometry (LC–ESI–MS/MS) produces large sets of MS/MS spectra. There is a need to assess the quality of large sets of experimental results by statistical comparison with that of random expectation. Classical frequency-based statistics such as goodness-of-fit tests for peptide-to-protein distributions could be used to calculate the probability that an entire set of experimental results has arisen by random chance. The frequency distributions of authentic MS/MS spectra from human blood were compared with those of false positive MS/MS spectra generated by a computer, or instrument noise, using the chi-square test. Here the mechanics of the chi-square test to compare the results in toto from a set of LC–ESI–MS/MS experiments with those of random expectation is detailed. The chi-square analysis of authentic spectra demonstrates unambiguously that the analysis of blood proteins separated by partition chromatography prior to tryptic digestions has a low probability that the cumulative peptide-to-protein distribution is the same as that of random or noise false positive spectra. [Copyright &y& Elsevier]

Eph receptors are a family of receptor tyrosine kinases that control directional cell movement during various biological processes, including embryogenesis, neuronal pathfinding, and tumor formation. The biochemical pathways of Eph receptors are context-dependent in part because of the varied composition of a heterotypic, oligomeric, active Eph receptor complex. Downstream of the Eph receptors, little is known about the essential phosphorylation events that define the context and instruct cell movement. Here, we define a pathway that is required for Eph receptor B2 (EphB2)-mediated cell sorting and is conserved among multiple Eph receptors. Utilizing a HEK293 model of EphB2+/ephrinB1+ cell segregation, we found that the scaffold adaptor protein SH2 domain-containing adaptor protein B (Shb) is essential for EphB2 functionality. Further characterization revealed that Shb interacts with known modulators of cytoskeletal rearrangement and cell mobility, including Nck adaptor protein (Nck), p120-Ras GTPase-activating protein (RasGAP), and the α- and β-Chimaerin Rac GAPs. We noted that phosphorylation of Tyr297, Tyr246, and Tyr336 of Shb is required for EphB2-ephrinB1 boundary formation, as well as binding of Nck, RasGAP, and the chimaerins, respectively. Similar complexes were formed in the context of EphA4, EphA8, EphB2, and EphB4 receptor activation. These results indicate that phosphotyrosine-mediated signaling through Shb is essential in EphB2-mediated heterotypic cell segregation and suggest a conserved function for Shb downstream of multiple Eph receptors. [ABSTRACT FROM AUTHOR]

The human monocyte cell line U937 was differentiated into an adherent macrophage phenotype using phorbol 12-myristate 13-acetate (PMA) to assay the phagocytosis of oxidized low-density lipoprotein (oxLDL) that may play a role in atherosclerosis. Microbeads were coated with the inflammatory ligand oxLDL to create a novel phagocytosis assay that models the binding of macrophages to oxLDL in the solid phase such as found in the fatty streaks of the arteries. The oxLDL was prepared with LDL from human ethylenediaminetetraacetic acid (EDTA) plasma oxidized with an excess (5 mM) of the strong oxidizing agent CuSO 4 and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis with Western blot. The binding of the oxLDL to the beads was confirmed by DilC18–oxLDL staining and confocal microscopy in addition to trypsin digestion of the microbeads for liquid chromatography, electrospray ionization, and tandem mass spectrometry. Phagocytosis of the oxLDL versus human bulk immunoglobulin G1 (IgG1)-coated microbeads was assayed over time, in the presence and absence of serum factors, by pulse chase and with enzyme inhibitor treatments. The ligand beads were then stained with specific antibodies to oxLDL versus human IgG to differentially stain external versus engulfed ligand microbeads. The phagocytosis of oxLDL and IgG ligand microbeads was abolished by the actin polymerization inhibitors cytochalasin D and latrunculin. Pharmacological inhibitors of the receptor enzymes JAK, SRC, and PLC prevented both IgG and oxLDL receptor function. In contrast, the function of the oxLDL phagocytic receptor complex was more sensitive to inhibition of PTK2, PKC, and SYK activity. [ABSTRACT FROM AUTHOR]

Summary: Cell signaling depends on dynamic protein-protein interaction (PPI) networks, often assembled through modular domains each interacting with multiple peptide motifs. This complexity raises a conceptual challenge, namely to define whether a particular cellular response requires assembly of the complete PPI network of interest or can be driven by a specific interaction. To address this issue, we designed variants of the Grb2 SH2 domain (“pY-clamps”) whose specificity is highly biased toward a single phosphotyrosine (pY) motif among many potential pYXNX Grb2-binding sites. Surprisingly, directing Grb2 predominantly to a single pY site of the Ptpn11/Shp2 phosphatase, but not other sites tested, was sufficient for differentiation of the essential primitive endoderm lineage from embryonic stem cells. Our data suggest that discrete connections within complex PPI networks can underpin regulation of particular biological events. We propose that this directed wiring approach will be of general utility in functionally annotating specific PPIs. [ABSTRACT FROM AUTHOR]

Summary: Metazoan evolution involves increasing protein domain complexity, but how this relates to control of biological decisions remains uncertain. The Ras guanine nucleotide exchange factor (RasGEF) Sos1 and its adaptor Grb2 are multidomain proteins that couple fibroblast growth factor (FGF) signaling to activation of the Ras-Erk pathway during mammalian development and drive embryonic stem cells toward the primitive endoderm (PrE) lineage. We show that the ability of Sos1/Grb2 to appropriately regulate pluripotency and differentiation factors and to initiate PrE development requires collective binding of multiple Sos1/Grb2 domains to their protein and phospholipid ligands. This provides a cooperative system that only allows lineage commitment when all ligand-binding domains are occupied. Furthermore, our results indicate that the interaction domains of Sos1 and Grb2 have evolved so as to bind ligands not with maximal strength but with specificities and affinities that maintain cooperativity. This optimized system ensures that PrE lineage commitment occurs in a timely and selective manner during embryogenesis. [Copyright &y& Elsevier]

Summary: Vascular endothelial growth factor and its receptors, FLK1/KDR and FLT1, are key regulators of angiogenesis. Unlike FLK1/KDR, the role of FLT1 has remained elusive. FLT1 is produced as soluble (sFLT1) and full-length isoforms. Here, we show that pericytes from multiple tissues produce sFLT1. To define the biologic role of sFLT1, we chose the glomerular microvasculature as a model system. Deletion of Flt1 from specialized glomerular pericytes, known as podocytes, causes reorganization of their cytoskeleton with massive proteinuria and kidney failure, characteristic features of nephrotic syndrome in humans. The kinase-deficient allele of Flt1 rescues this phenotype, demonstrating dispensability of the full-length isoform. Using cell imaging, proteomics, and lipidomics, we show that sFLT1 binds to the glycosphingolipid GM3 in lipid rafts on the surface of podocytes, promoting adhesion and rapid actin reorganization. sFLT1 also regulates pericyte function in vessels outside of the kidney. Our findings demonstrate an autocrine function for sFLT1 to control pericyte behavior. [ABSTRACT FROM AUTHOR]

Summary Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1. Graphical Abstract Highlights • Treatment with JQ1 induces an extensive BET proteins interactome rewiring • Structural and biophysical studies expand the target space for BET bromodomains • Two distinct short linear motifs mediate BET ET domain interactions • BRD3 negatively regulates proliferation through Pol I and II mechanisms Lambert, Picaud, et al. report that pharmacological bromodomain inhibition rewires the interactome of the Bromo and Extra-Terminal (BET) proteins, resulting in loss (e.g., histones), maintenance, or gain of interactions. They reveal new binding modalities and an unsuspected negative role for BRD3 in proliferation. [ABSTRACT FROM AUTHOR]