Anti-Peptide Monoclonal Antibodies Generated for Immuno-Multiple Reaction Monitoring-Mass Spectrometry Assays Have a High Probability of Supporting Western blot and ELISA

Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents because commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose analytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium''s (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first determination of the success rate (92%) for generating mAbs for immuno-MRM using a recombinant B cell cloning approach, which is considerably faster than the traditional hybridoma approach.The ability to measure specific proteins of interest is critical to the basic sciences and clinical research. To this end, immunoaffinity-based assays such as Western blotting, immunohistochemistry, and ELISAs have been in use for decades, but have several shortcomings including difficulty in multiplexing, a lack of standardization, and a semi-quantitative nature (e.g. Western blotting and immunohistochemistry) (1). Recently, there has been tremendous growth in using the sensitive, specific, multiplexable, and quantitative technology, multiple reaction monitoring-mass spectrometry, to measure tryptic peptides as stoichiometric surrogates for the detection of proteins from complex samples (2–7). The sensitivity of targeted multiple reaction monitoring (MRM)¹ is enhanced 10³–10⁴-fold by coupling it upstream with immunoaffinity enrichment of tryptic peptides in a peptide immuno-MRM assay (8–14). Advantages of immuno-MRM include high specificity, multiplexability (15, 16), and standardization, enabling high inter-laboratory reproducibility (17).The extent to which antibodies generated for immuno-MRM could support widely-used conventional immunoassay formats has not been investigated. This question is important because a lack of validated affinity reagents is a major obstacle to widespread implementation of immuno-MRM, which has considerable analytical advantages over traditional methods. Because the market for immuno-MRM is at present small relative to that for widely adopted conventional immunoassay formats (e.g. Western blotting and ELISA), commercial antibody suppliers are not incentivized to develop content specifically for immuno-MRM assays. Thus, we reasoned that if antibodies could be generated that are capable of supporting both conventional technologies as well as the emerging MRM platform, this might spark commercial interest by increasing the value of the antibodies, ultimately providing reagents to foster widespread implementation of immuno-MRM.Antigens used for antibody generation in conventional assays typically consist of either purified proteins, protein segments of 100–150 amino acids, or synthetic peptide sequences (18, 19). Antigenic prediction algorithms are often used to identify regions of target proteins that are most likely to be exposed on the surface of the protein and, thus, accessible for antibody binding. In contrast, proteotypic peptide antigens are selected for development of antibodies for immuno-MRM based on their uniqueness in the genome and their robust detectability by mass spectrometry, without regard to protein structure (because the protein will be proteolyzed during the assay). Because some widely used conventional immunoassay formats (e.g. Western blotting and indirect ELISA) detect proteins in their denatured form, it was reasonable to ask whether antibodies raised against short, linear, tryptic peptides would also work in these alternative formats.Here, we develop, characterize, and make publicly available 40 novel immuno-MRM assays and the associated monoclonals, and report the success rate of generating recombinant monoclonal antibodies (mAbs) that work in immuno-MRM assays. Furthermore, we determine the cross-over success rates of applying the mAbs in Western blotting and indirect ELISA assays.     

Vibrissa Self-Motion and Touch Are Reliably Encoded along the Same Somatosensory Pathway from Brainstem through Thalamus

Active sensing involves the fusion of internally generated motor events with external sensation. For rodents, active somatosensation includes scanning the immediate environment with the mystacial vibrissae. In doing so, the vibrissae may touch an object at any angle in the whisk cycle. The representation of touch and vibrissa self-motion may in principle be encoded along separate pathways, or share a single pathway, from the periphery to cortex. Past studies established that the spike rates in neurons along the lemniscal pathway from receptors to cortex, which includes the principal trigeminal and ventral-posterior-medial thalamic nuclei, are substantially modulated by touch. In contrast, spike rates along the paralemniscal pathway, which includes the rostral spinal trigeminal interpolaris, posteromedial thalamic, and ventral zona incerta nuclei, are only weakly modulated by touch. Here we find that neurons along the lemniscal pathway robustly encode rhythmic whisking on a cycle-by-cycle basis, while encoding along the paralemniscal pathway is relatively poor. Thus, the representations of both touch and self-motion share one pathway. In fact, some individual neurons carry both signals, so that upstream neurons with a supralinear gain function could, in principle, demodulate these signals to recover the known decoding of touch as a function of vibrissa position in the whisk cycle.     

AAV-Delivered Antibody Mediates Significant Protective Effects against SIVmac239 Challenge in the Absence of Neutralizing Activity

Long-term delivery of potent broadly-neutralizing antibodies is a promising approach for the prevention of HIV-1 infection. We used AAV vector intramuscularly to deliver anti-SIV monoclonal antibodies (mAbs) in IgG1 form to rhesus monkeys. Persisting levels of delivered mAb as high as 270 μg/ml were achieved. However, host antibody responses to the delivered antibody were observed in 9 of the 12 monkeys and these appeared to limit the concentration of delivered antibody that could be achieved. This is reflected in the wide range of delivered mAb concentrations that were achieved: 1–270 μg/ml. Following repeated, marginal dose, intravenous challenge with the difficult-to-neutralize SIVmac239, the six monkeys in the AAV-5L7 IgG1 mAb group showed clear protective effects despite the absence of detectable neutralizing activity against the challenge virus. The protective effects included: lowering of viral load at peak height; lowering of viral load at set point; delay in the time to peak viral load from the time of the infectious virus exposure. All of these effects were statistically significant. In addition, the monkey with the highest level of delivered 5L7 mAb completely resisted six successive SIVmac239 i.v. challenges, including a final challenge with a dose of 10 i.v. infectious units. Our results demonstrate the continued promise of this approach for the prevention of HIV-1 infection in people. However, the problem of anti-antibody responses will need to be understood and overcome for the promise of this approach to be effectively realized.     

Lipidomic Analysis Links Mycobactin Synthase K to Iron Uptake and Virulence in M. tuberculosis

The prolonged survival of Mycobacterium tuberculosis (M. tb) in the host fundamentally depends on scavenging essential nutrients from host sources. M. tb scavenges non-heme iron using mycobactin and carboxymycobactin siderophores, synthesized by mycobactin synthases (Mbt). Although a general mechanism for mycobactin biosynthesis has been proposed, the biological functions of individual mbt genes remain largely untested. Through targeted gene deletion and global lipidomic profiling of intact bacteria, we identify the essential biochemical functions of two mycobactin synthases, MbtK and MbtN, in siderophore biosynthesis and their effects on bacterial growth in vitro and in vivo. The deletion mutant, ΔmbtN, produces only saturated mycobactin and carboxymycobactin, demonstrating an essential function of MbtN as the mycobactin dehydrogenase, which affects antigenicity but not iron uptake or M. tb growth. In contrast, deletion of mbtK ablated all known forms of mycobactin and its deoxy precursors, defining MbtK as the essential acyl transferase. The mbtK mutant showed markedly reduced iron scavenging and growth in vitro. Further, ΔmbtK was attenuated for growth in mice, demonstrating a non-redundant role of hydroxamate siderophores in virulence, even when other M. tb iron scavenging mechanisms are operative. The unbiased lipidomic approach also revealed unexpected consequences of perturbing mycobactin biosynthesis, including extreme depletion of mycobacterial phospholipids. Thus, lipidomic profiling highlights connections among iron acquisition, phospholipid homeostasis, and virulence, and identifies MbtK as a lynchpin at the crossroads of these phenotypes.     

Protein O-Glucosyltransferase 1 (POGLUT1) Promotes Mouse Gastrulation through Modification of the Apical Polarity Protein CRUMBS2

Crumbs family proteins are apical transmembrane proteins with ancient roles in cell polarity. Mouse Crumbs2 mutants arrest at midgestation with abnormal neural plate morphology and a deficit of mesoderm caused by defects in gastrulation. We identified an ENU-induced mutation, wsnp, that phenocopies the Crumbs2 null phenotype. We show that wsnp is a null allele of Protein O-glucosyltransferase 1 (Poglut1), which encodes an enzyme previously shown to add O-glucose to EGF repeats in the extracellular domain of Drosophila and mammalian Notch, but the role of POGLUT1 in mammalian gastrulation has not been investigated. As predicted, we find that POGLUT1 is essential for Notch signaling in the early mouse embryo. However, the loss of mouse POGLUT1 causes an earlier and more dramatic phenotype than does the loss of activity of the Notch pathway, indicating that POGLUT1 has additional biologically relevant substrates. Using mass spectrometry, we show that POGLUT1 modifies EGF repeats in the extracellular domain of full-length mouse CRUMBS2. CRUMBS2 that lacks the O-glucose modification fails to be enriched on the apical plasma membrane and instead accumulates in the endoplasmic reticulum. The data demonstrate that CRUMBS2 is the target of POGLUT1 for the gastrulation epithelial-to-mesenchymal transitions (EMT) and that all activity of CRUMBS2 depends on modification by POGLUT1. Mutations in human POGLUT1 cause Dowling-Degos Disease, POGLUT1 is overexpressed in a variety of tumor cells, and mutations in the EGF repeats of human CRUMBS proteins are associated with human congenital nephrosis, retinitis pigmentosa and retinal degeneration, suggesting that O-glucosylation of CRUMBS proteins has broad roles in human health.     

Contribution of the Two Genes Encoding Histone Variant H3.3 to Viability and Fertility in Mice

Histones package DNA and regulate epigenetic states. For the latter, probably the most important histone is H3. Mammals have three near-identical H3 isoforms: canonical H3.1 and H3.2, and the replication-independent variant H3.3. This variant can accumulate in slowly dividing somatic cells, replacing canonical H3. Some replication-independent histones, through their ability to incorporate outside S-phase, are functionally important in the very slowly dividing mammalian germ line. Much remains to be learned of H3.3 functions in germ cell development.Histone H3.3 presents a unique genetic paradigm in that two conventional intron-containing genes encode the identical protein. Here, we present a comprehensive analysis of the developmental effects of null mutations in each of these genes. H3f3a mutants were viable to adulthood. Females were fertile, while males were subfertile with dysmorphic spermatozoa. H3f3b mutants were growth-deficient, dying at birth. H3f3b heterozygotes were also growth-deficient, with males being sterile because of arrest of round spermatids. This sterility was not accompanied by abnormalities in sex chromosome inactivation in meiosis I. Conditional ablation of H3f3b at the beginning of folliculogenesis resulted in zygote cleavage failure, establishing H3f3b as a maternal-effect gene, and revealing a requirement for H3.3 in the first mitosis. Simultaneous ablation of H3f3a and H3f3b in folliculogenesis resulted in early primary oocyte death, demonstrating a crucial role for H3.3 in oogenesis.These findings reveal a heavy reliance on H3.3 for growth, gametogenesis, and fertilization, identifying developmental processes that are particularly susceptible to H3.3 deficiency. They also reveal partial redundancy in function of H3f3a and H3f3b, with the latter gene being generally the most important.     

Immuno‐modification of enhancing stem cells targeting for myocardial repair

Despite the controversy in mechanism, rodent and clinical studies have demonstrated beneficial effects of stem/progenitor cell therapy after myocardial infarction (MI). In a rat ischaemic reperfusion MI model, we investigated the effects of immunomodification of CD 34⁺ cells on heart function and myocardial conduction. Bispecific antibody (BiAb), consisting of an anti‐myosin light chain antibody and anti‐CD45 antibody, injected intravenously was used to direct human CD34⁺ cells to injured myocardium. Results were compared to echocardiography guided intramyocardial (IM) injection of CD34⁺ cells and PBS injected intravenously. Treatment was administered 2 days post MI. Echocardiography was performed at 5 weeks and 3 months which demonstrated LV dilatation prevention and fractional shortening improvement in both the BiAb and IM injection approaches, with BiAb achieving better results. Histological analyses demonstrated a decrease in infarct size and increase in arteriogenesis in both BiAb and IM injection. Electrophysiological properties were studied 5 weeks after treatments by optical mapping. Conduction velocity (CV), action potential duration (APD) and rise time were significantly altered in the MI area. The BiAb treated group demonstrated a more normalized activation pattern of conduction and normalization of CV at shorter pacing cycle lengths. The ventricular tachycardia inducibility was lowest in the BiAb treatment group. Intravenous administration of BiAb offers an effective means of stem cell delivery for myocardial repair post‐acute MI. Such non‐invasive approach was shown to offer a distinct advantage to more invasive direct IM delivery.     

Building the Foundation for International Conservation Planning for Breeding Ducks across the U.S. and Canadian Border

We used publically available data on duck breeding distribution and recently compiled geospatial data on upland habitat and environmental conditions to develop a spatially explicit model of breeding duck populations across the entire Prairie Pothole Region (PPR). Our spatial population models were able to identify key areas for duck conservation across the PPR and predict between 62.1 – 79.1% (68.4% avg.) of the variation in duck counts by year from 2002 – 2010. The median difference in observed vs. predicted duck counts at a transect segment level was 4.6 ducks. Our models are the first seamless spatially explicit models of waterfowl abundance across the entire PPR and represent an initial step toward joint conservation planning between Prairie Pothole and Prairie Habitat Joint Ventures. Our work demonstrates that when spatial and temporal variation for highly mobile birds is incorporated into conservation planning it will likely increase the habitat area required to support defined population goals. A major goal of the current North American Waterfowl Management Plan and subsequent action plan is the linking of harvest and habitat management. We contend incorporation of spatial aspects will increase the likelihood of coherent joint harvest and habitat management decisions. Our results show at a minimum, it is possible to produce spatially explicit waterfowl abundance models that when summed across survey strata will produce similar strata level population estimates as the design-based Waterfowl Breeding Pair and Habitat Survey (r² = 0.977). This is important because these design-based population estimates are currently used to set duck harvest regulations and to set duck population and habitat goals for the North American Waterfowl Management Plan. We hope this effort generates discussion on the important linkages between spatial and temporal variation in population size, and distribution relative to habitat quantity and quality when linking habitat and population goals across this important region.     

Multi-Parametric MRI at 14T for Muscular Dystrophy Mice Treated with AAV Vector-Mediated Gene Therapy

The objective of this study was to investigate the efficacy of using quantitative magnetic resonance imaging (MRI) as a non-invasive tool for the monitoring of gene therapy for muscular dystrophy. The clinical investigations for this family of diseases often involve surgical biopsy which limits the amount of information that can be obtained due to the invasive nature of the procedure. Thus, other non-invasive tools may provide more opportunities for disease assessment and treatment responses. In order to explore this, dystrophic mdx^4cv mice were systemically treated with a recombinant adeno-associated viral (AAV) vector containing a codon-optimized micro-dystrophin gene. Multi-parametric MRI of T2, magnetization transfer, and diffusion effects alongside 3-D volume measurements were then utilized to monitor disease/treatment progression. Mice were imaged at 10 weeks of age for pre-treatment, then again post-treatment at 8, 16, and 24 week time points. The efficacy of treatment was assessed by physiological assays for improvements in function and quantification of expression. Tissues from the hindlimbs were collected for histological analysis after the final time point for comparison with MRI results. We found that introduction of the micro-dystrophin gene restored some aspects of normal muscle histology and pathology such as decreased necrosis and resistance to contraction-induced injury. T2 relaxation values showed percentage decreases across all muscle types measured (tibialis anterior, gastrocnemius, and soleus) when treated groups were compared to untreated groups. Additionally, the differences between groups were statistically significant for the tibialis anterior as well. The diffusion measurements showed a wider range of percentage changes and less statistical significance while the magnetization transfer effect measurements showed minimal change. MR images displayed hyper-intense regions of muscle that correlated with muscle pathology in histological sections. T2 relaxation, alongside diffusion and magnetization transfer effects provides useful data towards the goal of non-invasively monitoring the treatment of muscular dystrophy.