Evaluation of Parameters for Confident Phosphorylation Site Localization using an Orbitrap Fusion Tribrid Mass Spectrometer

Confident identification of sites of protein phosphorylation by mass spectrometry (MS) is essential to advance understanding of phosphorylation-mediated signaling events. However, development of novel instrumentation requires that methods for MS data acquisition and its interrogation be evaluated and optimized for high throughput phosphoproteomics. Here, we compare and contrast eight MS acquisition methods on the novel tribrid Orbitrap Fusion MS platform, using both a synthetic phosphopeptide library and a complex phosphopeptide-enriched cell lysate. As well as evaluating multiple fragmentation regimes (HCD, EThcD and neutral loss triggered ET(ca/hc)D), and analyzers for MS/MS (orbitrap (OT) versus ion trap (IT)), we also compare two commonly used bioinformatics platforms, Andromeda with PTM-score, and MASCOT with ptmRS, for confident phosphopeptide identification and, crucially, phosphosite localization. Our findings demonstrate that optimal phosphosite identification is achieved using HCD fragmentation and high resolution orbitrap-based MS/MS analysis, employing MASCOT/ptmRS for data interrogation. Although EThcD is optimal for confident site localization for a given PSM, the increased duty cycle compared with HCD compromises the numbers of phosphosites identified. Finally, our data highlights that a charge-state dependent fragmentation regime, and a multiple algorithm search strategy, are likely to be of benefit for confident large-scale phosphosite localization

Local protein kinase A action proceeds through intact holoenzymes

Hormones can transmit signals through adenosine 3’,5’-monophosphate (cAMP) to precise intracellular locations. The fidelity of these responses relies on the activation of localized protein kinase A (PKA) holoenzymes. Association of PKA regulatory (RII) subunits with A-kinase anchoring proteins (AKAPs) confers location, and catalytic (C) subunits phosphorylate substrates. Single-particle electron microscopy demonstrated that AKAP79 constrains RII-C sub-assemblies within 150 to 250Å of its targets. Native mass spectrometry established that these macromolecular assemblies incorporated stoichiometric amounts of cAMP. Chemical-biology and live-cell imaging techniques revealed that catalytically active PKA holoenzymes remained intact within the cytoplasm. In contrast, little, if any PKA activity was detected in the nucleus. Hence the parameters of anchored PKA holoenzyme action are much more restricted than originally anticipated

KinView: A visual comparative sequence analysis tool for integrated kinome research

Multiple sequence alignments (MSAs) are a fundamental analysis tool used throughout biology to investigate relationships between protein sequence, structure, function, evolutionary history, and patterns of disease-associated variants. However, their widespread application in systems biology research is currently hindered by the lack of user-friendly tools to simultaneously visualize, manipulate and query the information conceptualized in large sequence alignments, and the challenges in integrating MSAs with multiple orthogonal data such as cancer variants and post-translational modifications, which are often stored in heterogeneous data sources and formats. Here, we present the Multiple Sequence Alignment Ontology (MSAOnt), which represents a profile or consensus alignment in an ontological format. Subsets of the alignment are easily selected through the SPARQL Protocol and RDF Query Language for downstream statistical analysis or visualization. We have also created the Kinome Viewer (KinView), an interactive integrative visualization that places eukaryotic protein kinase cancer variants in the context of natural sequence variation and experimentally determined post-translational modifications, which play central roles in the regulation of cellular signaling pathways. Using KinView, we identified differential phosphorylation patterns between tyrosine and serine/threonine kinases in the activation segment, a major kinase regulatory region that is often mutated in proliferative diseases. We discuss cancer variants that disrupt phosphorylation sites in the activation segment, and show how KinView can be used as a comparative tool to identify differences and similarities in natural variation, cancer variants and post-translational modifications between kinase groups, families and subfamilies. Based on KinView comparisons, we identify and experimentally characterize a regulatory tyrosine (Y177PLK4) in the PLK4 C-terminal activation segment region termed the P+1 loop. To further demonstrate the application of KinView in hypothesis generation and testing, we formulate and validate a hypothesis explaining a novel predicted loss-of-function variant (D523NPKCβ) in the regulatory spine of PKCβ, a recently identified tumor suppressor kinase. KinView provides a novel, extensible interface for performing comparative analyses between subsets of kinases and for integrating multiple types of residue specific annotations in user friendly formats

Annotation extensions

The specificity of knowledge that Gene Ontology (GO) annotations currently can represent is still restricted by the legacy format of the GO annotation file, a format intentionally designed for simplicity to keep the barriers to entry low and thus encourage initial adoption. Historically, the information that could be captured in a GO annotation was simply the role or location of a gene product, although genetically interacting or binding partners could be specified. While there was no mechanism within the original GO annotation format for capturing additional information about the context of a GO term, such as the target gene of an activity or the location of a molecular function, the long-term vision for the GO Consortium was to provide greater expressivity in its annotations to capture physiologically relevant information. Thus, as a step forwards, the GO Consortium has introduced a new field into the annotation format, annotation extensions, which can be used to capture valuable contextual detail. This provides experimentally verified links between gene products and other physiological information that is crucial for accurate analysis of pathway and network data. This chapter will provide a simple overview of annotation extensions, illustrated with examples of their usage, and explain why they are useful for scientists and bioinformaticians alike

DOSCATs: Double standards for protein quantification

The two most common techniques for absolute protein quantification are based on either mass spectrometry (MS) or on immunochemical techniques, such as western blotting (WB). Western blotting is most often used for protein identification or relative quantification, but can also be deployed for absolute quantification if appropriate calibration standards are used. MS based techniques offer superior data quality and reproducibility, but WB offers greater sensitivity and accessibility to most researchers. It would be advantageous to apply both techniques for orthogonal quantification, but workflows rarely overlap. We describe DOSCATs (DOuble Standard conCATamers), novel calibration standards based on QconCAT technology, to unite these platforms. DOSCATs combine a series of epitope sequences concatenated with tryptic peptides in a single artificial protein to create internal tryptic peptide standards for MS as well as an intact protein bearing multiple linear epitopes. A DOSCAT protein was designed and constructed to quantify five proteins of the NF-κB pathway. For three target proteins, protein fold change and absolute copy per cell values measured by MS and WB were in excellent agreement. This demonstrates that DOSCATs can be used as multiplexed, dual purpose standards, readily deployed in a single workflow, supporting seamless quantitative transition from MS to WB

Covalent Aurora A regulation by the metabolic integrator coenzyme A

Aurora A kinase is a master mitotic regulator whose functions are controlled by several regulatory interactions and post-translational modifications. It is frequently dysregulated in cancer, making Aurora A inhibition a very attractive antitumor target. However, recently uncovered links between Aurora A, cellular metabolism and redox regulation are not well understood. In this study, we report a novel mechanism of Aurora A regulation in the cellular response to oxidative stress through CoAlation. A combination of biochemical, biophysical, crystallographic and cell biology approaches revealed a new and, to our knowledge, unique mode of Aurora A inhibition by CoA, involving selective binding of the ADP moiety of CoA to the ATP binding pocket and covalent modification of Cys290 in the activation loop by the thiol group of the pantetheine tail. We provide evidence that covalent CoA modification (CoAlation) of Aurora A is specific, and that it can be induced by oxidative stress in human cells. Oxidising agents, such as diamide, hydrogen peroxide and menadione were found to induce Thr 288 phosphorylation and DTT-dependent dimerization of Aurora A. Moreover, microinjection of CoA into fertilized mouse embryos disrupts bipolar spindle formation and the alignment of chromosomes, consistent with Aurora A inhibition. Altogether, our data reveal CoA as a new, rather selective, inhibitor of Aurora A, which locks this kinase in an inactive state via a “dual anchor” mechanism of inhibition that might also operate in cellular response to oxidative stress. Finally and most importantly, we believe that these novel findings provide a new rationale for developing effective and irreversible inhibitors of Aurora A, and perhaps other protein kinases containing appropriately conserved Cys residues

The p38/MK2/Hsp25 Pathway Is Required for BMP-2-Induced Cell Migration

Background: Bone morphogenetic proteins (BMPs) have been shown to participate in the patterning and specification of several tissues and organs during development and to regulate cell growth, differentiation and migration in different cell types. BMP-mediated cell migration requires activation of the small GTPase Cdc42 and LIMK1 activities. In our earlier report we showed that activation of LIMK1 also requires the activation of PAKs through Cdc42 and PI3K. However, the requirement of additional signaling is not clearly known. Methodology/Principal Findings: Activation of p38 MAPK has been shown to be relevant for a number of BMP-2¿s physiological effects. We report here that BMP-2 regulation of cell migration and actin cytoskeleton remodelling are dependent on p38 activity. BMP-2 treatment of mesenchymal cells results in activation of the p38/MK2/Hsp25 signaling pathway downstream from the BMP receptors. Moreover, chemical inhibition of p38 signaling or genetic ablation of either p38¿ or MK2 blocks the ability to activate the downstream effectors of the pathway and abolishes BMP-2-induction of cell migration. These signaling effects on p38/MK2/Hsp25 do not require the activity of either Cdc42 or PAK, whereas p38/MK2 activities do not significantly modify the BMP-2-dependent activation of LIMK1, measured by either kinase activity or with an antibody raised against phospho-threonine 508 at its activation loop. Finally, phosphorylated Hsp25 colocalizes with the BMP receptor complexes in lamellipodia and overexpression of a phosphorylation mutant form of Hsp25 is able to abolish the migration of cells in response to BMP-2. Conclusions: These results indicate that Cdc42/PAK/LIMK1 and p38/MK2/Hsp25 pathways, acting in parallel and modulating specific actin regulatory proteins, play a critical role in integrating responses during BMP-induced actin reorganization and cell migration

Intermittent preventive treatment for malaria in pregnancy in Africa: What's new, what's needed?

Falciparum malaria is an important cause of maternal, perinatal and neonatal morbidity in high transmission settings in Sub-Saharan Africa. Intermittent preventive treatment with sulphadoxine-pyrimethamine (SP-IPT) has proven efficacious in reducing the burden of pregnancy-associated malaria but increasing levels of parasite resistance mean that the benefits of national SP-IPT programmes may soon be seriously undermined in much of the region. Hence, there is an urgent need to develop alternative drug regimens for IPT in pregnancy. This paper reviews published safety and efficacy data on various antimalarials and proposes several candidate combination regimens for assessment in phase II/III clinical trials