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.     

The kinetics of the hypoferraemic response and changes in levels of alternative complement activity in diploid and triploid Atlantic salmon, following injection of lipopolysaccharide.

To study any possible effects of triploidy on the kinetics of the response of two non-specific disease factors, full sibling diploid and triploid Atlantic salmon were injected intraperitoneally with either lipopolysaccharide (1 mg kg(-1) body weight) or saline. Individually marked fish were repetitively blood sampled for up to 19 days. Total serum protein concentrations remained constant throughout the experiment indicating that the sampling regime did not cause haemodilution. The alternative complement pathway activity (measured by the titre of haemolytic activity against rabbit erythrocytes) in the serum of saline injected fish remained constant but in LPS-injected fish it fell to barely detectable levels 2 days after injection, but recovered to pre-treatment levels by about day 5. Triploid fish took slightly longer to reach full recovery levels than diploids. All groups of fish showed a hypoferraemic response, suggesting that the sampling regime was at least partially responsible. However, the response was more rapid and pronounced in the LPS-injected fish. In the latter, serum iron concentrations decreased to very low levels by day 2 post-injection in the diploid fish and by day 3 in the triploid fish. Pre-treatment iron levels were re-established by about 15 days post-injection in all groups. The data show only slight differences between the diploid and triploid fish, but the longer time taken for the triploids to recover complement activity and the slower onset of the hypoferraemic response following injection of LPS, suggest that they may be at a disadvantage compared with their diploid siblings in their defence against bacterial infections.     

The design and discovery of novel amide CCR5 antagonists

The synthesis of a range of novel amine-containing structures and their primary potency as inhibitors of HIV-1 fusion via blocking of the CCR5 receptor is described. The development of the medicinal chemistry strategy and SAR’s which led to the identification of the piperidine amide compounds 33 and 36 as excellent leads for further evaluation is described, along with key physicochemical data which highlighted their lead potential.     

Evaluation of creatinine phosphokinase in screening patients for malignant hyperpyrexia.

Evidence is presented that serum creatinine phosphokinase (CPK) activity is of no direct value in screening patients for susceptibility to malignant hyperpyrexia and does not correlate with halothane-induced muscle contracture or the presence of myopathy. Widely differing CPK values were found at different times in the same people. In most "malignant hyperpyrexia" families the susceptible patients had either normal or inconsistently raised CPK values.     

Evolution of ruminant Sarcocystis (Sporozoa) parasites based on small subunit rDNA sequences

We present an evolutionary analysis of 13 species of Sarcocystis, including 4 newly sequenced species with ruminants as their intermediate host, based on complete small subunit rDNA sequences. Those species with ruminants as their intermediate host form a well-supported clade, and there are at least two major clades within this group, one containing those species forming microcysts and with dogs as their definitive host and the other containing those species forming macrocysts and with cats as their definitive host. Those species with nonruminants as their intermediate host form the paraphyletic sister group to these clades. Most of the species have considerable genotypic differences (differing in more than 100 nucleotide positions), except for S. buffalonis and S. hirsuta. There is a large suite of genotypic differences indicating that those species infecting ruminant and nonruminant hosts have had very different evolutionary histories, and similarly for the felid- and canid-infecting species. Furthermore, the rDNA sequences that represent the different structural regions of the rRNA molecule have very different genotypic behavior within Sarcocystis. The evolution of these regions should be functionally constrained, and their differences can be explained in terms of the importance of the nucleotide sequences to their functions.     

Stimulation of prostaglandin-dependent macrophage suppressor cells by the subcutaneous injection of polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs), in the form of pure linoleic, linolenic, or arachidonic acid, were injected subcutaneously into male C57Bl/6 mice daily for 10 days. Injection of 3.6 mg/day of PUFA resulted in up to a two- to threefold increase in spleen weight. Spleen cell response to mitogens was reduced by about 70%; mixed lymphocyte responses were reduced by about 90% when compared to normal values. In admixture experiments, spleen cells from PUFA treated mice suppressed the mitogen induced blastogenic response of control spleen cells by up to 90%. Fractionation of spleen cells from PUFA treated mice by G-10 adherence resulted in an enrichment of suppressive activity in the adherent cells. The suppressive effect of G-10 adherent cells was abolished by the addition of indomethacin as well as by depletion of macrophages by treatments with agents such as carbonyl iron and leucine methyl ester. These studies indicate that the administration of PUFA has marked immunosuppressive effects in mice. These effects may be related to increased prostaglandin production and appear to be mediated by a macrophage type cell.     

Helicobacter Hypothesis for Idiopathic Parkinsonism: Before and Beyond

We challenge the concept of idiopathic parkinsonism (IP) as inevitably progressive neurodegeneration, proposing a natural history of sequential microbial insults with predisposing host response. Proof-of-principle that infection can contribute to IP was provided by case studies and a placebo-controlled efficacy study of Helicobacter eradication. "Malignant" IP appears converted to "benign", but marked deterioration accompanies failure. Similar benefit on brady/hypokinesia from eradicating "low-density" infection favors autoimmunity. Although a minority of UK probands are urea breath test positive for Helicobacter , the predicted probability of having the parkinsonian label depends on the serum H. pylori antibody profile, with clinically relevant gradients between this "discriminant index" and disease burden and progression. In IP, H. pylori antibodies discriminate for persistently abnormal bowel function, and specific abnormal duodenal enterocyte mitochondrial morphology is described in relation to H. pylori infection. Slow intestinal transit manifests as constipation from the prodrome. Diarrhea may flag secondary small-intestinal bacterial overgrowth. This, coupled with genetically determined intense inflammatory response, might explain evolution from brady/hypokinetic to rigidity-predominant parkinsonism.     

Expression of engrailed proteins in arthropods, annelids, and chordates

engrailed is a homeobox gene that has an important role in Drosophila segmentation. Genes homologous to engrailed have been identified in several other organisms. Here we describe a monoclonal antibody that recognizes a conserved epitope in the homeodomain of engrailed proteins of a number of different arthropods, annelids, and chordates; we use this antibody to isolate the grasshopper engrailed gene. In Drosophila embryos, the antibody reveals engrailed protein in the posterior portion of each segment during segmentation, and in a segmentally reiterated subset of neuronal cells during neurogenesis. Other arthropods, including grasshopper and two crustaceans, have similar patterns of engrailed expression. However, these patterns of expression are not shared by the annelids or chordates we examined. Our results provide the most comprehensive view that has been obtained of how expression patterns of a regulatory gene vary during evolution. On the basis of these patterns, we suggest that engrailed is a gene whose ancestral function was in neurogenesis and whose function was co-opted during the evolution of segmentation in the arthropods, but not in the annelids and chordates.     

Kinetics of juvenile sea bass (Dicentrarchus labrax, L.) systemic and mucosal antibody secreting cell response to different antigens (Photobacterium damselae spp. piscicida, Vibrio anguillarum and DNP).

The ELISPOT assay was used to measure the number of specific antibody secreting cells (ASC) induced during the primary and secondary immune responses in the spleen, head kidney and gut of juvenile (5 g) sea bass (Dicentrarchus labrax) to bacterial (Vibrio anguillarum and Photobacterium damselae ssp. piscicida) and hapten dinitrophenyl-conjugated to keyhole limpet haemocyanin (DNP-KLH) antigens administered intraperitoneally. High variability among individuals was observed at each sampling day. All fish were bath vaccinated to V. anguillarum at an earlier stage (2 g) in the farm of origin prior to the development of the experiments, and therefore only secondary and tertiary responses were measured in the group immunised with this bacterium. Significant differences to the controls were observed in the primary responses of the head kidney and the spleen to P. damselae ssp. piscicida and DNP, respectively. Frequency analysis of the production of ASC suggests that significant responses in the gut might be masked by the high error variance. The peak of the primary response was observed 4 days earlier to DNP (18-20 days post-immunisation) and it was significantly higher than the response to P. damselae ssp. piscicida. Higher numbers of ASC were observed in the secondary responses of the head kidney and spleen, although they were not statistically significantly different from the primary levels, probably due to the high error variance as supported by the frequency analysis. Nevertheless, together with a faster response (peak at 7 days post-immunisation), the data suggest that memory formation had occurred. Additionally, the data suggest that some suppression of the secondary immune response in the gut might have occurred. The head kidney appears to produce the highest number of specific ASC of the organs tested. It appears that sea bass show a relatively fast but short duration antibody response.