capture-compound-mass-spectrometry-powerful-tool-to-identify-novel-c

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

The assembly of synapses and neuronal circuits relies on an array of molecular recognition events and their modification by neuronal activity. Neurexins are a highly polymorphic family of synaptic receptors diversified by extensive alternative splicing. Neurexin variants exhibit distinct isoform-specific biochemical interactions and synapse assembly functions, but the mechanisms governing splice isoform choice are not understood. We demonstrate that Nrxn1 alternative splicing is temporally and spatially controlled in the mouse brain. Neuronal activity triggers a shift in Nrxn1 splice isoform choice via calcium/calmodulin-dependent kinase IV signaling. Activity-dependent alternative splicing of Nrxn1 requires the KH-domain RNA-binding protein SAM68 that associates with RNA response elements in the Nrxn1 pre-mRNA. Our findings uncover SAM68 as a key regulator of dynamic control of Nrxn1 molecular diversity and activity-dependent alternative splicing in the central nervous system.

SAM68 regulates neuronal activity-dependent alternative splicing of neurexin-1.

Protein complexes represent major functional units for the execution of biological processes. Systematic affinity purification coupled with mass spectrometry (AP-MS) yielded a wealth of information on the compendium of protein complexes expressed in Saccharomyces cerevisiae. However, global AP-MS analysis of human protein complexes is hampered by the low throughput, sensitivity and data robustness of existing procedures, which limit its application for systems biology research. Here, we address these limitations by a novel integrated method, which we applied and benchmarked for the human protein phosphatase 2A system. We identified a total of 197 protein interactions with high reproducibility, showing the coexistence of distinct classes of phosphatase complexes that are linked to proteins implicated in mitosis, cell signalling, DNA damage control and more. These results show that the presented analytical process will substantially advance throughput and reproducibility in future systematic AP-MS studies on human protein complexes.

An integrated workflow for charting the human interaction proteome: insights into the PP2A system.

We present a mass spectrometry-based strategy for the absolute quantification of protein complex components isolated through affinity purification. We quantified bait proteins via isotope-labeled reference peptides corresponding to an affinity tag sequence and prey proteins by label-free correlational quantification using the precursor ion signal intensities of proteotypic peptides generated in reciprocal purifications. We used this method to quantitatively analyze interaction stoichiometries in the human protein phosphatase 2A network.

Quantitative interaction proteomics using mass spectrometry.

Rift Valley fever virus (RVFV) continues to cause large outbreaks of acute febrile and often fatal illness among humans and domesticated animals in Africa, Saudi Arabia, and Yemen. The high pathogenicity of this bunyavirus is mainly due to the viral protein NSs, which was shown to prevent transcriptional induction of the antivirally active type I interferons (alpha/beta interferon [IFN-alpha/beta]). Viruses lacking the NSs gene induce synthesis of IFNs and are therefore attenuated, whereas the noninducing wild-type RVFV strains can only be inhibited by pretreatment with IFN. We demonstrate here in vitro and in vivo that a substantial part of the antiviral activity of IFN against RVFV is due to a double-stranded RNA-dependent protein kinase (PKR). PKR-mediated virus inhibition, however, was much more pronounced for the strain Clone 13 with NSs deleted than for the NSs-expressing strain ZH548. In vivo, Clone 13 was nonpathogenic for wild-type (wt) mice but could regain pathogenicity if mice lacked the PKR gene. ZH548, in contrast, killed both wt and PKR knockout mice indiscriminately. ZH548 was largely resistant to the antiviral properties of PKR because RVFV NSs triggered the specific degradation of PKR via the proteasome. The NSs proteins of the related but less virulent sandfly fever Sicilian virus and La Crosse virus, in contrast, had no such anti-PKR activity despite being efficient suppressors of IFN induction. Our data suggest that RVFV NSs has gained an additional anti-IFN function that may explain the extraordinary pathogenicity of this virus.

NSs protein of rift valley fever virus induces the specific degradation of the double-stranded RNA-dependent protein kinase.

Proliferation of mammalian cells requires the coordinated function of many proteins to accurately divide a cell into two daughter cells. Several RNAi screens have identified previously uncharacterised genes that are implicated in mammalian cell division. The molecular function for these genes needs to be investigated to place them into pathways. Phenotypic profiling is a useful method to assign putative functions to uncharacterised genes. Here, we show that the analysis of protein localisation is useful to refine a phenotypic profile. We show the utility of this approach by defining a function of the previously uncharacterised gene C13orf3 during cell division. C13orf3 localises to centrosomes, the mitotic spindle, kinetochores, spindle midzone, and the cleavage furrow during cell division and is specifically phosphorylated during mitosis. Furthermore, C13orf3 is required for centrosome integrity and anaphase onset. Depletion by RNAi leads to mitotic arrest in metaphase with an activation of the spindle assembly checkpoint and loss of sister chromatid cohesion. Proteomic analyses identify C13orf3 (Ska3) as a new component of the Ska complex and show a direct interaction with a regulatory subunit of the protein phosphatase PP2A. All together, these data identify C13orf3 as an important factor for metaphase to anaphase progression and highlight the potential of combined RNAi screening and protein localisation analyses.

Comparative profiling identifies C13orf3 as a component of the Ska complex required for mammalian cell division.

Verbreitung der S&auml;ugetier-Zellen erfordert die koordinierte Funktion vieler Proteine genau eine Zelle in zwei Tochterzellen unterteilen. Mehrere RNAi-Bildschirme haben zuvor uncharacterised Gene identifiziert, die in S&auml;ugetieren Zellteilung verwickelt sind. Die molekulare Funktion f&uuml;r diese Gene muss untersucht werden, um sie in Bahnen zu platzieren. Ph&auml;notypische profiling ist eine n&uuml;tzliche Methode uncharacterised Gene vermeintliche Funktionen zuweisen. Hier zeigen wir, dass die Analyse von Protein-Lokalisierung eine ph&auml;notypische Profil verfeinern kann. Wir zeigen das Dienstprogramm dieses Ansatzes durch die Definition einer Funktion im zuvor uncharacterised gen C13orf3 w&auml;hrend der Zellteilung. C13orf3 dynamisches Centrosomes, die mitotische Spindel, Kinetochores, Spindel Midzone und die Trennungsfurche w&auml;hrend der Zellteilung und ist speziell w&auml;hrend der Mitose phosphoryliert. Dar&uuml;ber hinaus ist die C13orf3 f&uuml;r Zentrosom Integrit&auml;t und Anaphase Beginn erforderlich. Ersch&ouml;pfung durch RNAi f&uuml;hrt zur mitotischen Verhaftung in Metaphase mit einer Aktivierung der Spindel Baugruppe Pr&uuml;fpunkt und Verlust der Schwester Chromatid Zusammenhalt. Proteomic Analysen identifizieren C13orf3 (Ska3) als neue Komponente der Ska-komplex und zeigen eine direkte Interaktion mit einem regulatorischen Untereinheit von den Protein-Phosphatase PP2A. Alle zusammen, diese Daten C13orf3 als ein wichtiger Faktor f&uuml;r Metaphase Anaphase Fortschreiten zu identifizieren und markieren Sie das Potenzial der kombinierten RNAi-Screening und Protein-Lokalisierung-Analysen.

Vergleichende Profilerstellung bezeichnet C13orf3 als Bestandteil der komplexen Ska f&uuml;r S&auml;ugetier-Zellteilung ben&ouml;tigt.

The AAA-ATPase VCP (also known as p97) cooperates with distinct cofactors to process ubiquitylated proteins in different cellular pathways. VCP missense mutations cause a systemic degenerative disease in humans, but the molecular pathogenesis is unclear. We used an unbiased mass spectrometry approach and identified a VCP complex with the UBXD1 cofactor, which binds to the plasma membrane protein caveolin-1 (CAV1) and whose formation is specifically disrupted by disease-associated mutations. We show that VCP-UBXD1 targets mono-ubiquitylated CAV1 in SDS-resistant high-molecular-weight complexes on endosomes, which are en route to degradation in endolysosomes. Expression of VCP mutant proteins, chemical inhibition of VCP, or siRNA-mediated depletion of UBXD1 leads to a block of CAV1 transport at the limiting membrane of enlarged endosomes in cultured cells. In patient muscle, muscle-specific caveolin-3 accumulates in sarcoplasmic pools and specifically delocalizes from the sarcolemma. These results extend the cellular functions of VCP to mediating sorting of ubiquitylated cargo in the endocytic pathway and indicate that impaired trafficking of caveolin may contribute to pathogenesis in individuals with VCP mutations.

Endolysosomal sorting of ubiquitylated caveolin-1 is regulated by VCP and UBXD1 and impaired by VCP disease mutations.

Genetic analysis in Drosophila melanogaster has been widely used to identify a system of genes that control cell growth in response to insulin and nutrients. Many of these genes encode components of the insulin receptor/target of rapamycin (InR/TOR) pathway. However, the biochemical context of this regulatory system is still poorly characterized in Drosophila. Here, we present the first quantitative study that systematically characterizes the modularity and hormone sensitivity of the interaction proteome underlying growth control by the dInR/TOR pathway. Applying quantitative affinity purification and mass spectrometry, we identified 97 high confidence protein interactions among 58 network components. In all, 22% of the detected interactions were regulated by insulin affecting membrane proximal as well as intracellular signaling complexes. Systematic functional analysis linked a subset of network components to the control of dTORC1 and dTORC2 activity. Furthermore, our data suggest the presence of three distinct dTOR kinase complexes, including the evolutionary conserved dTTT complex (Drosophila TOR, TELO2, TTI1). Subsequent genetic studies in flies suggest a role for dTTT in controlling cell growth via a dTORC1- and dTORC2-dependent mechanism.

Modularity and hormone sensitivity of the Drosophila melanogaster insulin receptor/target of rapamycin interaction proteome.

We present a statistical method SAINT-MS1 for scoring protein-protein interactions based on the label-free MS1 intensity data from affinity purification-mass spectrometry (AP-MS) experiments. The method is an extension of Significance Analysis of INTeractome (SAINT), a model-based method previously developed for spectral count data. We reformulated the statistical model for log-transformed intensity data, including adequate treatment of missing observations, that is, interactions identified in some but not all replicate purifications. We demonstrate the performance of SAINT-MS1 using two recently published data sets: a small LTQ-Orbitrap data set with three replicate purifications of single human bait protein and control purifications and a larger drosophila data set targeting insulin receptor/target of rapamycin signaling pathway generated using an LTQ-FT instrument. Using the drosophila data set, we also compare and discuss the performance of SAINT analysis based on spectral count and MS1 intensity data in terms of the recovery of orthologous and literature-curated interactions. Given rapid advances in high mass accuracy instrumentation and intensity-based label-free quantification software, we expect that SAINT-MS1 will become a useful tool allowing improved detection of protein interactions in label-free AP-MS data, especially in the low abundance range.

SAINT-MS1: protein-protein interaction scoring using label-free intensity data in affinity purification-mass spectrometry experiments.

The second messenger cyclic di-GMP is a near-ubiquitous signaling molecule that globally alters bacterial cell physiology to promote biofilm formation and community behavior. Much progress was made in recent years towards the identification and characterization of diguanylate cyclases and phosphodiersterases, enzymes involved in the synthesis and degradation of this signaling compound. In contrast, our knowledge of the nature and mechanistic details of c-di-GMP effector proteins lags behind, primarily because effective tools for their specific enrichment and rapid analysis are missing. In this report we demonstrate that a novel tri-functional c-di-GMP-specific Capture Compound (cdG-CC) can be effectively used to identify and validate c-di-GMP binding proteins. The cdG-CC was able to specifically and efficiently pull down bona fide c-di-GMP effector proteins. Furthermore, in combination with mass spectrometry (CCMS), this technology robustly identified a substantial fraction of the known c-di-GMP signaling components directly from cell extracts of different model organisms. Finally, we applied the CCMS technique to profile c-di-GMP binding proteins of Pseudomonas aeruginosa and Salmonella enterica serovar typhimurium. Our studies establish CCMS as a powerful and versatile tool to identify and analyze components of the cellular c-di-GMP pathway in a wide range of different organisms.

A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins.

The complete and specific proteolytic cleavage of protein samples into peptides is crucial for the success of every shotgun LC-MS/MS experiment. In particular, popular peptide-based label-free and targeted mass spectrometry approaches rely on efficient generation of fully cleaved peptides to ensure accurate and sensitive protein quantification. In contrast to previous studies, we globally and quantitatively assessed the efficiency of different digestion strategies using a yeast cell lysate, label-free quantification, and statistical analysis. Digestion conditions include double tryptic, surfactant-assisted, and tandem-combinatorial Lys-C/trypsin digestion. In comparison to tryptic digests, Lys-C/trypsin digests were found most efficient to yield fully cleaved peptides while reducing the abundance of miscleaved peptides. Subsequent sequence context analysis revealed improved digestion performances of Lys-C/trypsin for miscleaved sequence stretches flanked by charged basic and particulary acidic residues. Furthermore, targeted MS analysis demonstrated a more comprehensive protein cleavage only after Lys-C/trypsin digestion, resulting in a more accurrate absolute protein quantification and extending the number of peptides suitable for SRM assay development. Therefore, we conclude that a serial Lys-C/trypsin digestion is highly attractive for most applications in quantitative MS-based proteomics building on in-solution digestion schemes.

Large-scale quantitative assessment of different in-solution protein digestion protocols reveals superior cleavage efficiency of tandem Lys-C/trypsin proteolysis over trypsin digestion.

Topoisomerase I (TOP1) inhibitors are an important class of anticancer drugs. The cytotoxicity of TOP1 inhibitors can be modulated by replication fork reversal through a process that requires poly(ADP-ribose) polymerase (PARP) activity. Whether regressed forks can efficiently restart and what factors are required to restart fork progression after fork reversal are still unknown. We have combined biochemical and EM approaches with single-molecule DNA fiber analysis to identify a key role for human RECQ1 helicase in replication fork restart after TOP1 inhibition that is not shared by other human RecQ proteins. We show that the poly(ADP-ribosyl)ation activity of PARP1 stabilizes forks in the regressed state by limiting their restart by RECQ1. These studies provide new mechanistic insights into the roles of RECQ1 and PARP in DNA replication and offer molecular perspectives to potentiate chemotherapeutic regimens based on TOP1 inhibition.

Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition.

There is a great interest in reliable ways to obtain absolute protein abundances at a proteome-wide scale. To this end, label-free LC-MS/MS quantification methods have been proposed where all identified proteins are assigned an estimated abundance. Several variants of this quantification approach have been presented, based on either the number of spectral counts per protein or MS1 peak intensities. Equipped with several datasets representing real biological environments, containing a high number of accurately quantified reference proteins, we evaluate five popular low-cost and easily implemented quantification methods (Absolute Protein Expression, Exponentially Modified Protein Abundance Index, Intensity-Based Absolute Quantification Index, Top3, and MeanInt). Our results demonstrate considerably improved abundance estimates upon implementing accurately quantified reference proteins; that is, using spiked in stable isotope labeled standard peptides or a standard protein mix, to generate a properly calibrated quantification model. We show that only the Top3 method is directly proportional to protein abundance over the full quantification range and is the preferred method in the absence of reference protein measurements. Additionally, we demonstrate that spectral count based quantification methods are associated with higher errors than MS1 peak intensity based methods. Furthermore, we investigate the impact of miscleaved, modified, and shared peptides as well as protein size and the number of employed reference proteins on quantification accuracy.

Critical assessment of proteome-wide label-free absolute abundance estimation strategies.

AMPylation is a posttranslational modification (PTM) that has recently caught much attention in the context of bacterial infections as pathogens were shown to secrete Fic proteins that AMPylate Rho GTPases and thus interfere with host cell signaling processes. Although Fic proteins are widespread and found in all kingdoms of life, only a small number of AMPylation targets are known to date. A major obstacle to target identification is the limited availability of generic strategies allowing sensitive and robust identification of AMPylation events. Here, we present an unbiased MS-based approach utilizing stable isotope-labeled ATP. The ATP isotopes are transferred onto target proteins in crude cell lysates by in vitro AMPylation introducing specific reporter ion clusters that allow detection of AMPylated peptides in complex biological samples by MS analysis. Applying this strategy on the secreted Fic protein Bep2 of Bartonella rochalimae, we identified the filamenting protein vimentin as an AMPylation target that was confirmed by independent assays. Vimentin represents a new class of target proteins and its identification emphasizes our method as a valuable tool to systematically uncover AMPylation targets. Furthermore, the approach can be generically adapted to study targets of other PTMs that allow incorporation of isotopically labeled substrates.

An experimental strategy for the identification of AMPylation targets from complex protein samples.

The ability of Salmonella to cause disease depends on metabolic activities and virulence factors. Here, we show that a key metabolic protein, EIIAGlc, is absolutely essential for acute infection, but not for Salmonella survival, in a mouse typhoid fever model. Surprisingly, phosphorylation-dependent EIIAGlc functions, including carbohydrate transport and activation of adenylate cyclase for global regulation, do not explain this virulence phenotype. Instead, biochemical studies, in vitro secretion and translocation assays, and in vivo genetic epistasis experiments suggest that EIIAGlc binds to the type three secretion system 2 (TTSS-2) involved in systemic virulence, stabilizes its cytoplasmic part including the crucial TTSS-2 ATPase, and activates virulence factor secretion. This unexpected role of EIIAGlc reveals a striking direct link between central Salmonella metabolism and a crucial virulence mechanism.

The central metabolism regulator EIIAGlc switches Salmonella from growth arrest to acute virulence through activation of virulence factor secretion.

In recent years, directed and, particularly, targeted mass spectrometric workflows have gained momentum as alternative techniques to conventional data-dependent acquisition (DDA) LC-MS/MS approaches. By focusing on specific peptide species, these methods allow hypothesis-driven analysis of selected proteins of interest, and they have been shown to be suited to monitor low-abundance proteins within complex mixtures. Despite their growing popularity, no study has systematically evaluated these various MS strategies in terms of quantification, detection, and identification limits when they are applied to complex samples. Here, we systematically compared the performance of conventional DDA, directed, and various targeted MS approaches on two different instruments, namely, a hybrid linear ion trap--Orbitrap and a triple quadrupole instrument. We assessed the limits of identification, quantification, and detection for each method by analyzing a dilution series of 20 unmodified and 10 phosphorylated synthetic heavy-labeled reference peptides, respectively, covering 6 orders of magnitude in peptide concentration with and without a complex human cell digest background. We found that all methods performed similarly in the absence of background proteins; however, when analyzing whole-cell lysates, targeted methods were at least 5-10 times more sensitive than that of the directed or DDA method. In particular, higher stage fragmentation (MS3) of the neutral loss peak using a linear ion trap increased the dynamic quantification range of some phosphopeptides up to 100-fold. We illustrate the power of this targeted MS3 approach for phosphopeptide monitoring by successfully quantifying nine phosphorylation sites of the kinetochore and spindle assembly checkpoint component Mad1 over different cell cycle states from nonenriched pull-down samples.

Evaluation of data-dependent and -independent mass spectrometric workflows for sensitive quantification of proteins and phosphorylation sites.

Centrioles function as core components of centrosomes and as basal bodies for the formation of cilia and flagella. Thus, effective control of centriole numbers is essential for embryogenesis, tissue homeostasis, and genome stability. In mammalian cells, the centriole duplication cycle is governed by Polo-like kinase 4 (Plk4). Here we identify the E3 ubiquitin ligase Mind bomb (Mib1) as a novel interaction partner of Plk4. We show that Mib1 localizes to centriolar satellites but redistributes to centrioles in response to conditions that induce centriole amplification. The E3 ligase activity of Mib1 triggers ubiquitination of Plk4 on multiple sites, causing the formation of Lys11-, Lys29- and Lys48-ubiquitin linkages. These modifications control the abundance of Plk4 and its ability to interact with centrosomal proteins, thus counteracting centriole amplification induced by excess Plk4. Collectively, these results identify the interaction between Mib1 and Plk4 as a novel important element in the control of centriole homeostasis.

Article	Year
SAM68 regulates neuronal activity-dependent alternative splicing of neurexin-1. Cell\| PubMed ID: 22196734	2011
An integrated workflow for charting the human interaction proteome: insights into the PP2A system. Molecular systems biology\| PubMed ID: 19156129	2009
Quantitative interaction proteomics using mass spectrometry. Nature methods\| PubMed ID: 19198594	2009
NSs protein of rift valley fever virus induces the specific degradation of the double-stranded RNA-dependent protein kinase. Journal of virology\| PubMed ID: 19211744	2009
Comparative profiling identifies C13orf3 as a component of the Ska complex required for mammalian cell division. The EMBO journal\| PubMed ID: 19387489	2009
Endolysosomal sorting of ubiquitylated caveolin-1 is regulated by VCP and UBXD1 and impaired by VCP disease mutations. Nature cell biology\| PubMed ID: 21822278	2011
Modularity and hormone sensitivity of the Drosophila melanogaster insulin receptor/target of rapamycin interaction proteome. Molecular systems biology\| PubMed ID: 22068330	2011
SAINT-MS1: protein-protein interaction scoring using label-free intensity data in affinity purification-mass spectrometry experiments. Journal of proteome research\| PubMed ID: 22352807	2012
A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins. Journal of proteomics\| PubMed ID: 22652488	2012
Large-scale quantitative assessment of different in-solution protein digestion protocols reveals superior cleavage efficiency of tandem Lys-C/trypsin proteolysis over trypsin digestion. Journal of proteome research\| PubMed ID: 23017020	2012
Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition. Nature structural & molecular biology\| PubMed ID: 23396353	2013
Critical assessment of proteome-wide label-free absolute abundance estimation strategies. Proteomics\| PubMed ID: 23794183	2013
An experimental strategy for the identification of AMPylation targets from complex protein samples. Proteomics\| PubMed ID: 24677795	2014
The central metabolism regulator EIIAGlc switches Salmonella from growth arrest to acute virulence through activation of virulence factor secretion. Cell reports\| PubMed ID: 24835993	2014
Evaluation of data-dependent and -independent mass spectrometric workflows for sensitive quantification of proteins and phosphorylation sites. Journal of proteome research\| PubMed ID: 25330945	2014
The E3 ubiquitin ligase Mib1 regulates Plk4 and centriole biogenesis. Journal of cell science\| PubMed ID: 25795303	2015

Timo Glatter

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