High-throughput Quantitative Approach Research Articles

12-Plex DiLeu Isobaric Labeling Enabled High-Throughput Investigation of Citrullination Alterations in the DNA Damage Response.

Protein citrullination is a key post-translational modification (PTM) that leads to the loss of positive charge on arginine and consequent protein structural and functional changes. Though it has been indicated to play critical roles in various physiological and pathological processes, effective analytical tools are largely limited due to a few challenges such as the small mass shift induced by this PTM and its low-abundance nature. Recently, we developed a biotin thiol tag, which enabled large-scale profiling of protein citrullination from complex biological samples via mass spectrometry. However, a high-throughput quantitative approach is still in great need to further improve the understanding of this PTM. In this study, we report an efficient pipeline using our custom-developed N,N-dimethyl leucine isobaric tags to achieve a multiplexed quantitative analysis of citrullination from up to 12 samples for the first time. We then apply this strategy to investigating citrullination alterations in response to DNA damage stress using human cell lines. We unveil important biological functions regulated by protein citrullination and observe hypercitrullination on RNA-binding proteins and DNA repair proteins, respectively. Our results reveal the involvement of citrullination in DNA damage pathways and may provide new insights into DNA-damage-related disease pathogenesis.

Proteomics of Protein Trafficking

Organs are interdependent and specialized – they communicate to enable organismal processes including growth, energy metabolism, and reproduction. Secreted peptides and proteins are essential regulators of homeostasis, and they compose a poorly-characterized communication network. Secreted factors are also relevant for disease: obesity affects 40% of the US population and results from poorly-characterized dysfunctional systemic signaling. Despite their relevance to homeostasis and disease, many additional factors remain to be identified. Characterizing the factors involved has been challenging, because conventional methods cannot identify low-abundance proteins or their origins and destinations. The broad goal of my new laboratory at Scripps is to apply novel high-throughput quantitative approaches we developed to interorgan communication in mammals, in order to understand the identity of the factors involved, which organs they originate from and target, their functions, and regulation of their production in disease. In this ASBMB Annual Meeting talk, I will describe our recent work and progress in establishing a proteomic platform, in which we biotinylate all proteins in a subcellular compartment of one organ using the engineered promiscuous biotin ligase BirA*G3 (a relative of TurboID), and then affinity-enrich the biotinylated proteins, identify them from distant organs using quantitative proteomics. This platform therefore allows simplifying the proteome and identifying low-abundance proteins, as well as their organs of origin and destination. Using this system in Drosophila, we identified 269 fat body secreted proteins in muscles, which included growth factors, cytokines, and proteins with receptors in muscles. In particular, I will present the characterization of muscle phenotypes of a fat body-derived protein CG2145. In addition to these data, I will describe our ongoing work on transitioning this method to mammalian systems. Altogether, this approach should be widely applicable to studies of secretome trafficking networks in vivo, including non-conventional secretion, in both arthropod and mammalian systems, in healthy or diseased states.

Cell Kinetics in the Adult Neurogenic Niche and Impact of Diet-Induced Accelerated Aging

Neurogenesis in the adult brain, a powerful mechanism for neuronal plasticity and brain repair, is altered by aging and pathological conditions, including metabolic disorders. The search for mechanisms and therapeutic solutions to alter neurogenesis requires understanding of cell kinetics within neurogenic niches using a high-throughput quantitative approach. The challenge is in the dynamic nature of the process and multiple cell types involved, each having several potential modes of division or cell fate. Here we show that cell kinetics can be revealed through a combination of the BrdU/EdU pulse-chase, based on the circadian pattern of DNA replication, and a differential equations model that describes time-dependent cell densities. The model is validated through the analysis of cell kinetics in the cerebellar neurogenic niche of normal young adult male zebrafish, with cells quantified in 2D (sections), and with neuronal fate and reactivation of stem cells confirmed in 3D whole-brain images (CLARITY). We then reveal complex alterations in cell kinetics associated with accelerated aging due to chronic high caloric intake. Low activity of neuronal stem cells in this condition persists 2 months after reverting to normal diet, and is accompanied by overproduction of transient amplifying cells, their accelerated cell death, and slow migration of postmitotic progeny. This combined experimental and mathematical approach should allow for relatively high-throughput analysis of early signs of pathological and age-related changes in neurogenesis, evaluation of specific therapeutic targets, and drug efficacy.SIGNIFICANCE STATEMENT Understanding normal cell kinetics of adult neurogenesis and the type of cells affected by a pathological process is needed to develop effective prophylactic and therapeutic measures directed at specific cell targets. Complex time-dependent mechanisms involved in the kinetics of multiple cell types require a combination of experimental and mathematical modeling approaches. This study demonstrates such a combined approach by comparing normal neurogenesis with that altered by diet-induced accelerated aging in adult zebrafish.

Tandem In Situ Monitoring for Quantitative Assessment of Mechanochemical Reactions Involving Structurally Unknown Phases.

We report herein quantitative in situ monitoring by simultaneous PXRD and Raman spectroscopy of the mechanochemical reaction between benzoic acid and nicotinamide, affording a rich polymorphic system with four new cocrystal polymorphs, multiple phase transformations, and a variety of reaction pathways. After observing polymorphs by in situ monitoring, we were able to isolate and characterize three of the four polymorphs, most of which are not accessible from solution. Relative stabilities among the isolated polymorphs at ambient conditions were established by slurry experiments. Using two complementary methods for in situ monitoring enabled quantitative assessment and kinetic analysis of each studied mechanochemical reaction, even when involving unknown crystal structures, and short-lived intermediates. In situ Raman monitoring was introduced here also as a standalone laboratory technique for quantitative assessment of mechanochemical reactions and understanding of mechanochemical reactivity. Our results provide an important step toward a complete and high-throughput quantitative approach to mechanochemical reaction kinetics and mechanisms, necessary for the development of the mechanistic framework of milling reactions.

Large-scale microfluidic gradient arrays reveal axon guidance behaviors in hippocampal neurons

High-throughput quantitative approaches to study axon growth behaviors have remained a challenge. We have developed a 1024-chamber microfluidic gradient generator array that enables large-scale investigations of axon guidance and growth dynamics from individual primary mammalian neurons, which are exposed to gradients of diffusible molecules. Our microfluidic method (a) generates statistically rich data sets, (b) produces a stable, reproducible gradient with negligible shear stresses on the culture surface, (c) is amenable to the long-term culture of primary neurons without any unconventional protocol, and (d) eliminates the confounding influence of cell-secreted factors. Using this platform, we demonstrate that hippocampal axon guidance in response to a netrin-1 gradient is concentration-dependent—attractive at higher concentrations and repulsive at lower concentrations. We also show that the turning of the growth cone depends on the angle of incidence of the gradient. Our study highlights the potential of microfluidic devices in producing large amounts of data from morphogen and chemokine gradients that play essential roles not only in axonal navigation but also in stem cell differentiation, cell migration, and immune response.

A High-Throughput Quantitative Approach Reveals More Small RNA Modifications in Mouse Liver and Their Correlation with Diabetes

Studies of RNA modification are usually focused on tRNA. However the modification of other small RNAs, including 5.8S rRNA, 5S rRNA, and small RNA sized at 10-60 nt, is still largely unknown. In this study, we established an efficient method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously identify and quantify more than 40 different types of nucleosides in small RNAs. With this method, we revealed 23 modified nucleosides of tRNA from mouse liver, and 6 of them were observed for the first time in eukaryotic tRNA. Moreover, 5 and 4 modified nucleosides were detected for the first time in eukaryotic 5.8S and 5S rRNA, respectively, and 22 modified nucleosides were identified in the small RNAs sized at 30-60 or 10-30 nt. Interestingly, two groups of 5S rRNA peaks were observed when analyzed by HPLC, and the abundance of modified nucleosides is significantly different between the two groups of peaks. Further studies show that multiple modifications in small RNA from diabetic mouse liver are significantly increased or decreased. Taken together, our data revealed more modified nucleosides in various small RNAs and showed the correlation of small RNA modifications with diabetes. These results provide new insights to the role of modifications of small RNAs in their stability, biological functions, and correlation with diseases.

597: Metabolomic prediction of trisomy 18 first trimester pregnancies

first trimester pregnancies Ray Bahado-Singh, Ranjit Akolekar, Rupsari Mandal, Anushka Chelliah, Edison Dong, Jianguo Xia, Mike Kruger, David Wishart, Kypros NIcolaides Wayne State University School of Medicine, Obstetrics and Gynecology, Detroit, MI, Medway NHS Foundation Trust, Fetal Medicine Unit, Gillingham, United Kingdom, University of Alberta, Biological Sciences, Edmonton, AB, Canada, University of Alberta, Computing Sciences, Edmonton, AB, Canada, The Fetal Medicine Foundation, Fetal Medicine, London, United Kingdom OBJECTIVE: Metabolomics is a rapidly developing field of omics science that employs a high throughput quantitative approach to characterize small molecule metabolites and their metabolism at the system-wide level.. Our purpose was to determine whether the metabolomic profile is altered in Trisomy 18 (T18) pregnancies and whether these biomarkers can predict T18. STUDY DESIGN: This was a case control study. NMR based metabolomic analyses of maternal serum prospectively collected between 11 0 and 13 6 weeks were performed. Metabolite concentrations in 30 T18 and 114 euploid controls were compared. Statistical analyses including logistic regression and genetic programming (GP) [www.Thegmax.com] were performed. Predictive algorithms for T18 detection consisting of metabolites by themselves or in combination with maternal age and delta nuchal translucency ( NT) measurements were evaluated. RESULTS: Of a total of 40 metabolites analyzed, 29 had significant differences in serum concentrations between the groups. Several models for T18 prediction were developed. A logistic model-based on 2-hydroxybytrate, formate and maternal age had 56.7% sensitivity and 95.6 % specificity for T18 detection, AUC (95% CI); 0.91 (0.86, 0.96), p 0.001. For 2-hydroxybutyrate plus delta nuchal translucency ( NT) these values were 96.7% and 99.1%; AUC (95% CI): 0.99 (0.97Using GP analysis, a metabolite only model had 66.7% sensitivity and 99.1% specificity. The addition of NT further improved these values to 93.3% and 98.3 % respectively., 1.000), p 0.001 respectively. CONCLUSION: Conclusion: An extensive group difference in metabolomic profile was observed. Metabolomics appears to be a novel tool for aneuploidy prediction. When combined with NT measurements, preliminary data suggest that high diagnostic accuracy is achievable.

Gender-related differences in ion-channel and transporter subunit expression in non-diseased human hearts

Gender-related differences in ventricular electrophysiology are known to be important determinants of human arrhythmic risk, but the underlying molecular basis is poorly understood. The present work aims to provide the first detailed analysis of gender-related cardiac ion-channel gene-distribution, based on samples from non-diseased human hearts. By using a high-throughput quantitative approach, we investigated at a genome-scale the expression of 79 genes encoding ion-channel and transporter subunits in epicardial and endocardial tissue samples from non-diseased transplant donors (10 males, 10 females). Gender-related expression differences involved key genes implicated in conduction and repolarization. Female hearts showed reduced expression for a variety of K +-channel subunits with potentially important roles in cardiac repolarization, including HERG, minK, Kir2.3, Kv1.4, KChIP2, SUR2 and Kir6.2, as well as lower expression of connexin43 and phospholamban. In addition, they demonstrated an isoform switch in Na +/K +-ATPase, expressing more of the α1 and less of the α3 subunit than male hearts, along with increased expression of calmodulin-3. Iroquois transcription factors (IRX3, IRX5) were more strongly expressed in female than male epicardium, but the transmural gradient remained. Protein-expression paralleled transcript patterns for all subunits examined: HERG, minK, Kv1.4, KChIP2, IRX5, Nav1.5 and connexin43. Our results indicate that male and female human hearts have significant differences in ion-channel subunit composition, with female hearts showing decreased expression for a number of repolarizing ion-channels. These findings are important for understanding sex-related differences in the susceptibility to ventricular arrhythmias, particularly for conditions associated with repolarization abnormalities like Brugada and Long QT syndrome.

L004 New insights into the mechanisms of the cardiac electrophysiological changes with aging in mice

Age is the single biggest risk factor for the developing heart failure and other cardiac problems. With a large increase in the elderly population across Europe, and an associated large increase in elderly patients with chronic cardiac problems, the need to develop our understanding of the ageing process is more pressing than ever. By examining the ageing effect on the ion channel (IC) and transcriptional regulators (TR) expression of the heart, we gleaned novel information regarding the signalling potentially responsible for generating the electrophysiological remodelling with age. By using a high-throughput quantitative approach, we investigated at a genome-scale the expression of 78 genes encoding IC, transporter subunits and Ca2+-homeostasis molecules in ventricles from embryonic days E15.5, E18.5 and postnatal day 1, day 7, day 20 and adult C57BL6 mice. We have also mapped 158 TR transcripts in the same samples, combined with bioinformatic predictions of sites recognized by the TR and their targets. Among transcripts involved in electrical signalling and Ca2+-homeostasis, 26/78 exhibited up-regulated expression, while 24 showed down-regulated expression. Using one-way hierarchical clustering analysis, we identified the TRs similarly up- and down-regulated than the ion channel genes. We hypothesized that those clustered IC and TR genes are co-regulated and that they share cisregulatory elements. By in silico investigations, we predicted over-representation of: 1/ Essrα on Nav1.5, Kv2.1, HCN2, RYR2, KChIP2 and Cavα2δ1 promoters ; 2/ GATA5 and GATA6 on Cav3.2, Cav1.3, Cavα2δ2, NCX1, RIP3-2, Nedd4-2, Cx40 and Cx45 promoters, suggesting that these TRs are involved in the expression of these key ion channel for the cardiac electrical activity. These results were strengthened by inter-species conservation. Our results provide novel molecular correlates in the ageing heart. The biological validations of these data will give significant potential implications for understanding the mechanisms underlying the fœtal program gene re-expression in cardiac diseases.