Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling

A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks.Cell signaling research is faced with the challenging task of interrogating increasingly large numbers of analytes in “systems biology” approaches, while maintaining the high standards of integrity and reproducibility traditionally associated with the scientific approach. For example, studies interrogating complex systems, such as protein signaling networks, require quantification technologies capable of sensitive, specific, multiplexable, and reproducible application. However, recent reports have highlighted alarmingly high rates of irreproducibility in fundamental biological and pre-clinical studies (1, 2), as well as poor performance of affinity reagents used in traditional proteomic assay and detection platforms (3, 4). There is an imminent need for high quality assays, including highly characterized standards and detailed documentation of processes and procedures (5). To improve the translation of cell signaling discoveries into clinical application, we need reproducible and transferable technologies that enable higher throughput quantification of protein phosphorylation.Signaling dynamics through post-translational modifications (e.g. phosphorylation) are predominantly measured by Western blotting. Although this technique has led to many discoveries and is the de facto “gold standard,” it suffers from many drawbacks. Western blotting is a low throughput approach applied to individual analytes (i.e. no multiplexing) and is susceptible to erroneous interpretation when applied quantitatively (6). Alternative immunoassay platforms have emerged (e.g. immunohistochemistry, ELISA, mass cytometry, and bead-based or planar arrays), but suffer from similar limitations, namely specificity issues (because of cross-reactivity of antibodies), poor standardization, and difficulties in multiplexing.One alternative for quantifying phosphorylation is targeted, multiple reaction monitoring (MRM)¹ MS, a widely deployed technique in clinical laboratories for quantification of small molecules (7, 8). MRM is now also well established for precise and specific quantification of endogenous, proteotypic peptides relative to spiked-in stable isotope-labeled internal standards (9–11), and MRM can be applied to phosphopeptides (12–18). MRM assays can be run at high multiplex levels (19–21) and can be standardized to be highly reproducible across laboratories (22–24), even on an international stage (25). Because phosphorylation typically occurs at sub-stoichiometric levels and because phosphopeptides must compete for ionization with more abundant peptides, mass spectrometry-based analysis of phosphorylation requires an analyte enrichment step. Immuno-affinity enrichment approaches using anti-phospho-tyrosine antibodies (26) or panels of antibodies targeting signaling nodes (27) have been implemented with shotgun mass spectrometry. Although anti-peptide antibodies can also be used to enrich individual phosphopeptides upstream of MRM (28), the generation of these reagents is time-consuming and costly, limiting widespread uptake.Phosphopeptide enrichment based on metal affinity chromatography has recently matured into a reproducible approach (29). Immobilized metal affinity chromatography (IMAC) is widely used in discovery phosphoproteomic studies to enrich phosphopeptides upstream of shotgun-based mass spectrometry (30, 31). We hypothesized that a subset of the cellular phosphoproteome with favorable binding characteristics to the IMAC resin might be reproducibly recovered for quantification when coupled with quantitative MRM mass spectrometry, enabling robust IMAC-MRM assays without the need for an antibody.In this report, we: (1) demonstrate the feasibility of generating analytically robust, multiplex IMAC-MRM assays for quantifying cellular phospho-signaling, (2) present a semi-automated, 96-well format magnetic bead-based protocol for IMAC enrichment, (3) provide a catalogue of phosphopeptides that are highly amenable to IMAC-MRM quantification, and (4) make publicly available standard operating protocols (SOP) and fit-for-purpose analytical validation data for IMAC-MRM assays targeting 107 phospho-analytes, providing a community resource for study of the DNA damage response. The data suggest that the IMAC-MRM approach is generally applicable to signaling pathways, enabling wider interrogation of signaling networks.     

Acquired Antibody Responses against Plasmodium vivax Infection Vary with Host Genotype for Duffy Antigen Receptor for Chemokines (DARC)

Background

Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC) is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are ‘resistant’ to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens.

Methodology/Findings

We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1) and Duffy binding protein (PvDBP) varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull) were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B). The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion.

Conclusion/Significance

Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the primary mechanisms by which P. vivax evades host immunity is through DARC indirectly down-regulating humoral responses against erythrocytic invasion and development.     

Matrix quality and disturbance frequency drive evolution of species behavior at habitat boundaries

Previous theoretical studies suggest that a species' landscape should influence the evolution of its dispersal characteristics, because landscape structure affects the costs and benefits of dispersal. However, these studies have not considered the evolution of boundary crossing, that is, the tendency of animals to cross from habitat to nonhabitat (“matrix”). It is important to understand this dispersal behavior, because of its effects on the probability of population persistence. Boundary‐crossing behavior drives the rate of interaction with matrix, and thus, it influences the rate of movement among populations and the risk of dispersal mortality. We used an individual‐based, spatially explicit model to simulate the evolution of boundary crossing in response to landscape structure. Our simulations predict higher evolved probabilities of boundary crossing in landscapes with more habitat, less fragmented habitat, higher‐quality matrix, and more frequent disturbances (i.e., fewer generations between local population extinction events). Unexpectedly, our simulations also suggest that matrix quality and disturbance frequency have much stronger effects on the evolution of boundary crossing than either habitat amount or habitat fragmentation. Our results suggest that boundary‐crossing responses are most affected by the costs of dispersal through matrix and the benefits of escaping local extinction events. Evolution of optimal behavior at habitat boundaries in response to the landscape may have implications for species in human‐altered landscapes, because this behavior may become suboptimal if the landscape changes faster than the species' evolutionary response to that change. Understanding how matrix quality and habitat disturbance drive evolution of behavior at boundaries, and how this in turn influences the extinction risk of species in human‐altered landscapes should help us identify species of conservation concern and target them for management.     

A direct test of the effects of changing atmospheric pressure on the mating behavior of Drosophila melanogaster

Changes in weather can be catastrophic for small insects. As such, it would be highly adaptive for insects to be able to sense when a weather front is approaching and respond appropriately. While correlative and anecdotal evidence exists that flies behaviorally respond to changes in barometric pressure, which indicate variation in weather, a direct test has yet to be performed. Here, we subject multiple strains of Drosophila melanogaster to changes in barometric pressure within a hypobaric chamber and measure male courtship and female receptivity. Since this species has a long copulation duration, copulating when adverse weather is approaching could subject both males and females to potentially lethal conditions. As predicted, some flies reduced their mating activity when exposed to a change in pressure that indicated imminent adverse weather. Surprisingly, however, some flies instead increased their mating activity; the behavioral response depended upon the strain’s native population location and intra-population variation, demonstrating that there is genetic variation for the behavioral response. This indicates that flies are able to anticipate weather patterns and change their behavior depending on the barometric pressure they experience, but that the form of behavioral response varies both within and between populations.     

Genomic data reveal ancient microendemism in forest scorpions across the California Floristic Province

The California Floristic Province (CFP) in western North America is a globally significant biodiversity hotspot. Elucidating patterns of endemism and the historical drivers of this diversity has been an important challenge of comparative phylogeography for over two decades. We generated phylogenomic data using ddRADseq to examine genetic structure in Uroctonus forest scorpions, an ecologically restricted and dispersal‐limited organism widely distributed across the CFP north to the Columbia River. We coupled our genetic data with species distribution models (SDMs) to determine climatically suitable areas for Uroctonus both now and during the Last Glacial Maximum. Based on our analyses, Uroctonus is composed of two major genetic groups that likely diverged over 2 million years ago. Each of these groups itself contains numerous genetic groups that reveal a pattern of vicariance and microendemism across the CFP. Migration rates among these populations are low. SDMs suggest forest scorpion habitat has remained relatively stable over the last 21 000 years, consistent with the genetic data. Our results suggest tectonic plate rafting, mountain uplift, river drainage formation and climate‐induced habitat fragmentation have all likely played a role in the diversification of Uroctonus. The intricate pattern of genetic fragmentation revealed across a temporal continuum highlights the potential of low‐dispersing species to shed light on small‐scale patterns of biodiversity and the underlying processes that have generated this diversity in biodiversity hotspots.     

Hx, a novel fluorescent, minor groove and sequence specific recognition element: design, synthesis, and DNA binding properties of p-anisylbenzimidazole-imidazole/pyrrole-containing polyamides

With the aim of incorporating a recognition element that acts as a fluorescent probe upon binding to DNA, three novel pyrrole (P) and imidazole (I)-containing polyamides were synthesized. The compounds contain a p-anisylbenzimidazolecarboxamido (Hx) moiety attached to a PP, IP, or PI unit, giving compounds HxPP (2), HxIP (3), and HxPI (4), respectively. These fluorescent hybrids were tested against their complementary nonfluorescent, non-formamido tetraamide counterparts, namely, PPPP (5), PPIP (6), and PPPI (7) (cognate sequences 5'-AAATTT-3', 5'-ATCGAT-3', and 5'-ACATGT-3', respectively). The binding affinities for both series of polyamides for their cognate and noncognate sequences were ascertained by surface plasmon resonance (SPR) studies, which revealed that the Hx-containing polyamides gave binding constants in the 10(6) M(-1) range while little binding was observed for the noncognates. The binding data were further compared to the corresponding and previously reported formamido-triamides f-PPP (8), f-PIP (9), and f-PPI (10). DNase I footprinting studies provided additional evidence that the Hx moiety behaved similarly to two consecutive pyrroles (PP found in 5-7), which also behaved like a formamido-pyrrole (f-P) unit found in distamycin and many formamido-triamides, including 8-10. The biophysical characterization of polyamides 2-7 on their binding to the abovementioned DNA sequences was determined using thermal melts (ΔT(M)), circular dichroism (CD), and isothermal titration calorimetry (ITC) studies. Density functional calculations (B3LYP) provided a theoretical framework that explains the similarity between PP and Hx on the basis of molecular electrostatic surfaces and dipole moments. Furthermore, emission studies on polyamides 2 and 3 showed that upon excitation at 322 nm binding to their respective cognate sequences resulted in an increase in fluorescence at 370 nm. These low molecular weight polyamides show promise for use as probes for monitoring DNA recognition processes in cells.     

Kinetic analysis of IgG antibodies to beta-amyloid oligomers with surface plasmon resonance

Surface plasmon resonance was used to investigate the kinetics, affinity, and specificity of binding between anti-Aβ (beta-amyloid) IgG antibodies and oligomeric Aβ. Two factors were needed to accurately characterize the IgG binding kinetics. First, a bivalent model was necessary to properly fit the kinetic association and dissociation sensograms. Second, a high concentration of IgG was necessary to overcome a significant mass transport limitation that existed regardless of oligomer density on the sensor surface. Using high IgG concentrations and bivalent fits, consistent kinetic parameters were found at varying sensor surface ligand densities. A comparison of binding specificity, affinity, and kinetic flux between monoclonal and natural human anti-Aβ IgG antibodies revealed the following findings. First, monoclonal antibodies 6E10 and 4G8 single-site binding affinity is similar between Aβ oligomers and monomers. Second, natural human anti-Aβ IgG binding readily binds Aβ oligomers but does not bind monomers. Third, natural human anti-Aβ IgG binds Aβ oligomers with a higher affinity and kinetic flux than 6E10 and 4G8. Both the current analytical methodology and antibody binding profiles are important for advances in antibody drug development and kinetic biomarker applications for Alzheimer’s disease.     

Bilateral cleft lip and nasal repair

SUMMARY: The bilateral cleft lip and nasal repair has remained a challenging endeavor. Techniques have evolved to address concerns over unsatisfactory features and stigmata of the surgery. The authors present an approach to this complex clinical problem that modifies traditional repairs described by Millard and Manchester. The senior author (H.S.B.) has developed this technique with over 25 years of surgical experience dealing with the bilateral cleft lip. This staged lip and nasal repair provides excellent nasal projection, lip function, and aesthetic outcomes. Lip repair is performed at 3 months of age. Columellar lengthening is performed at approximately 18 months of age. A key component of this repair focuses on reconstruction of the central tubercle. A triangular prolabial dry vermilion flap is augmented by lateral lip vermilion flaps that include the profundus muscle of the orbicularis oris. This minimizes lateral lip segment sacrifice and provides improved central vermilion fullness, which is often deficient in traditional repairs. The authors present the surgical technique and examples of their clinical results.