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.     

IL-22 Restrains Tapeworm-Mediated Protection against Experimental Colitis via Regulation of IL-25 Expression

Interleukin (IL)-22, an immune cell-derived cytokine whose receptor expression is restricted to non-immune cells (e.g. epithelial cells), can be anti-inflammatory and pro-inflammatory. Mice infected with the tapeworm Hymenolepis diminuta are protected from dinitrobenzene sulphonic acid (DNBS)-induced colitis. Here we assessed expulsion of H. diminuta, the concomitant immune response and the outcome of DNBS-induced colitis in wild-type (WT) and IL-22 deficient mice (IL-22^-/-) ± infection. Interleukin-22^-/- mice had a mildly impaired ability to expel the worm and this correlated with reduced or delayed induction of TH2 immunity as measured by splenic and mesenteric lymph node production of IL-4, IL-5 and IL-13 and intestinal Muc-2 mRNA and goblet cell hyperplasia; in contrast, IL-25 increased in the small intestine of IL-22^-/- mice 8 and 12 days post-infection compared to WT mice. In vitro experiments revealed that H. diminuta directly evoked epithelial production of IL-25 that was inhibited by recombinant IL-22. Also, IL-10 and markers of regulatory T cells were increased in IL-22^-/- mice that displayed less DNBS (3 mg, ir. 72h)-induced colitis. Wild-type mice infected with H. diminuta were protected from colitis, as were infected IL-22^-/- mice and the latter to a degree that they were almost indistinguishable from control, non-DNBS treated mice. Finally, treatment with anti-IL-25 antibodies exaggerated DNBS-induced colitis in IL-22^-/- mice and blocked the anti-colitic effect of infection with H. diminuta. Thus, IL-22 is identified as an endogenous brake on helminth-elicited TH2 immunity, reducing the efficacy of expulsion of H. diminuta and limiting the effectiveness of the anti-colitic events mobilized following infection with H. diminuta in a non-permissive host.     

The Protective Association between Pet Ownership and Depression among Street-involved Youth: A Cross-sectional Study

Street-involved youth represent a particularly vulnerable subsection of the homeless population and are at increased risk of health problems, substance abuse, and depression. Qualitative research has demon- strated that animal companions help homeless youth cope with loneliness, are motivators for positive change, such as decreasing drug or alcohol use, provide unconditional love without judgement, and improve youths’ sense of health. To quantitatively investigate the association between depression and pet ownership among street-involved youth, a cross-sectional study was per- formed with a convenience sample of 189 street-involved youths who were surveyed in four cities in Ontario, Canada, 89 of whom were pet owners and 100 of whom were not. Logistic regression modelling found pet ownership to be negatively associated with depression in the study population (controlling for gender, regular use of drugs, and time since youth left home), with the odds of being depressed three times greater for youths who did not own pets. While pet ownership among street-involved youth has many liabilities, includ- ing impairing youths’ ability to access shelter, services, and housing and employment opportunities, companion animals may offer both physical and psychosocial benefits that youth have difficult attaining. This finding highlights the importance of increased awareness among youth service providers of the potential impacts of pet ownership for street-involved youth.     

Assigning alleles to different loci in amplifications of duplicated loci

Duplicated loci, for example those associated with major histocompatibility complex (MHC) genes, often have similar DNA sequences that can be coamplified with a pair of primers. This results in genotyping difficulties and inaccurate analyses. Here, we present a method to assign alleles to different loci in amplifications of duplicated loci. This method simultaneously considers several factors that may each affect correct allele assignment. These are the sharing of identical alleles among loci, null alleles, copy number variation, negative amplification, heterozygote excess or heterozygote deficiency, and linkage disequilibrium. The possible multilocus genotypes are extracted from the alleles for each individual and weighted to estimate the allele frequencies. The likelihood of an allele configuration is calculated and is optimized with a heuristic algorithm. Monte‐Carlo simulations and three empirical MHC data sets are used as examples to evaluate the efficacy of our method under different conditions. Our new software, mhc‐typer V1.1, is freely available at https://github.com/huangkang1987/mhc-typer .     

Invasion by activated macrophages requires delivery of nascent membrane‐type‐1 matrix metalloproteinase through late endosomes/lysosomes to the cell surface

Macrophage migration into injured or infected tissue is a key aspect in the pathophysiology of many diseases where inflammation is a driving factor. Membrane‐type‐1 matrix metalloproteinase (MT1‐MMP) cleaves extracellular matrix components to facilitate invasion. Here we show that, unlike the constitutive MT1‐MMP surface recycling seen in cancer cells, unactivated macrophages express low levels of MT1‐MMP. Upon lipopolysaccharide (LPS) activation, MT1‐MMP synthesis dramatically increases 10‐fold at the surface by 15 hours. MT1‐MMP is trafficked from the Golgi complex to the surface via late endosomes/lysosomes in a pathway regulated by the late endosome/lysosome R‐SNAREs VAMP7 and VAMP8. These form two separate complexes with the surface Q‐SNARE complex Stx4/SNAP23 to regulate MT1‐MMP delivery to the plasma membrane. Loss of either one of these SNAREs leads to a reduction in surface MT1‐MMP, gelatinase activity and reduced invasion. Thus, inhibiting MT1‐MMP transport through this pathway could reduce macrophage migration and the resulting inflammation.