ReactELISA: Monitoring a Carbon Nucleophilic ... - ACS Publications

Subscriber access provided by Van Pelt and Opie Library

Article

ReactELISA: Monitoring a Carbon Nucleophilic Metabolite by ELISA - a Study of Lipid Metabolism Emil Frank Holmquist, Ulrik Bering Keiding, Rasmus Kold-Christensen, Trine Salomón, Karl Anker Jørgensen, Peter Kristensen, Thomas B Poulsen, and Mogens Johannsen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00507 • Publication Date (Web): 04 Apr 2017 Downloaded from http://pubs.acs.org on April 9, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

ReactELISA: Monitoring a Carbon Nucleophilic Metabolite by ELISA - a Study of Lipid Metabolism Emil F. Holmquist,†‡ Ulrik B. Keiding,†‡ Rasmus Kold-Christensen, †‡ Trine Salomón,† Karl A. Jørgensen, ‡ Peter Kristensen,§ Thomas. B. Poulsen,‡ and Mogens Johannsen. †* †

Department of Forensic Medicine, Aarhus University, Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark

‡

Department of Chemistry, Aarhus University, Langelandsgade 140, 8000 Aarhus C, Denmark

§

Department of Engineering, Aarhus University, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark.

ABSTRACT: We here present a conceptually novel reaction-based ELISA principle (ReactELISA) for quantitation of the carbon nucleophilic lipid metabolite acetoacetate. Key to the assay is the utilization of a highly chemoselective Friedländer reaction that captures and simultaneously stabilizes the nucleophilic metabolite directly in the biological matrix. By developing a bifunctional biotinylated capture probe, the Friedländer-acetoacetate adduct can be trapped and purified directly in streptavidin coated wells. Finally, we outline the selection and refinement of a highly selective recombinant antibody for specific adduct quantitation. The setup is very robust and, as we demonstrate via miniaturization for microplate format, amenable for screening of compounds or interventions that alter lipid metabolism in liver cell cultures. The assay-principle should be extendable to quantitation of other nucleophilic or electrophilic and perhaps even more reactive metabolites provided suitable capture probes and antibodies.

Ketone bodies, acetoacetate (AcAcO) 1a and βhydroxybutyrate, function as cellular energy substitutes during periods of low carbohydrate availability, such as calorie restricted or ketogenic dieting, fasting or strenuous exercise, and their endogenous levels can increase more than 10-fold during such periods.1,2 Calorie restricted diets has for many years been known to confer protection against age-related diseases and to increase median life span in organisms ranging from yeast to primates.3,4 Initially, this was thought to be caused by the reduced intake of calories, however, more recent research indicates that enhanced lipo-oxidative metabolism and in particular the ketone bodies could be causally involved.5-9 Recent studies of longevity in mice and other organisms have also identified a few small molecules capable of extending life span, among these rapamycin and resveratrol.10,11 Though their mode of action is quite different, they share the common feature of enhancing lipid metabolism and ketogenesis.12,13 Furthermore ketone bodies, in particular AcAcO, are also

known for their dampening effect on intractable epileptic seizures as well as acting as general neuroprotective agents.14 These findings, as well as many other recent studies,15-20 has led to a growing interest in understanding the biological significance of ketone bodies, and highlight the pharmacological potential in discovering novel small molecules that enhance their formation. Small endogenous metabolites are inherently difficult to detect using conventional antibody based ELISAs, which is the routine method used in most biochemical laboratories.21,22 Alternatively, reliable quantification can be achieved by use of a derivatization reagent followed by more advanced instrumental detection by chromatography and mass spectrometry. This latter technique however requires specialized equipment and training23 and is not suited for high-throughput screening. Currently no ELISA for AcAcO exists. A colorimetric procedure is known based on diazonium reagents which

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

are used in excessive amounts.24 This method however lack

Figure 1. Principle of a ReactELISA. A Bifunctional probe is added to the biological matrix containing the reactive metabolite of interest. After reaction the probe/adduct mixture is purified by transfer to streptavidin coated 96-well plate, and washing away unbound matrix. The adduct is quantified by ELISA using an adduct-specific recombinant antibody.

specificity due to the formation of colored byproducts from reaction with tyrosine and oxaloacetate 1b and chromatography has to be applied to circumvent this issue.25 An indirect, but in principle specific enzymatic method using β-hydroxybutyrate dehydrogenase is also known,26 though in this method specificity can still be compromised by the presence of other endogenous oxidoreductases in e.g. plasma that also generate NADH/NAD+ which are used for quantitation.27 Here, we report a simple, robust, and specific reaction-based capture ELISA (ReactELISA) for quantification of AcAcO. This new detection-principle is based on bifunctional probes with a capture group and a biotin affinity tag. A chemoselective reaction between the capture group and the metabolite in a complex matrix initially generates a stable adduct. In the second step the adduct is trapped and purified using streptavidin-coated microtiter plates. The adduct is finally assayed by ELISA using an adductspecific recombinant antibody (Figure 1).28-29 By miniturization the format is ready for screening of small molecule perturbations of lipid metabolism.

EXPERIMENTAL SECTION For a detailed description of all protocols, procedures as well as syntheses see Supporting Information. General ReactELISA Protocol: Reaction conditions for adduct 13a formation. To a microtiter plate (Greiner Bio-one Cellstar clear 96-well plate, 300 µL) 180 µL biological samples were added 10 µL carbonate buffer (final pH plasma was 9.0 and culture media 9.75) and 10 µL 200 µM bifunctional probe 12. The 96-well plate was sealed with silicone seal (Thermo-Fischer Scientific) and incubated at 37 °C for 5 days (samples can after reaction be stored in the freezer until analysis). For cell experiments reaction volumes were scaled to 70 µL (see AML12 Hepatocyte Experiment section for specific conditions). Performing the reaction directly in the streptavidin plate used for the ELISA detection leads to poor results due to increased background from biological

Page 2 of 7

deposits from the plasma sample. ELISA analysis. 100 µL of reaction samples were transferred to a streptavidin coated microtiter plate (Pierce™ Streptavidin Coated High Capacity) and incubated at rt for 1 h. For experiments with a reaction volume less than 100 µL, 40 µL was transferred to a streptavidin coated microtiter plate containing 60 µL unreacted biological solution. After the incubation the samples were carefully removed followed by washing of the wells (5×300 µL 1×PBS). 100 µL blocking buffer (2 w/v % skimmed milk protein (Premier Foods) in PBS (MPBS)) was added and allowed to incubate for 2 h at rt or overnight at 4 °C. This blocking buffer was removed followed by a washing step (5×300 µL 1×PBS). 1.0 µg/mL adduct 13a specific antibody Ab1 (2% MPBS) was then added and allowed to incubate for 2 h at rt. After a washing step (3×300 µL 0.1 % PBST (0.1 % Tween 20 (Sigma Aldrich) in 1×PBS), 100 µL 1:4000 polyclonal swine anti-rabbit immunoglobulin-HRP (Dako) in 2% MPBS was added. The plate was incubated at rt for 1 h. Washing with 0.1 % PBST (3×300 µL) primed the plate for addition of 100 µL TMB single solution (Life Technologies). The TMB solution was allowed to react for 20 min (plasma) or 40 min (cell media) in complete darkness under two layers of tinfoil. In order to quench the reaction 50 µL 1 M H2SO4 was added and the plate was analyzed within 10 mins of the acid addition at OD450 subtracting OD655 (Tecan Spark 10M plate reader). UPLC-MS/MS analysis. In a 2 mL titer plate (Eppendorf) 200 µL of biological reaction samples were diluted with 100 µL Milli-Q H2O and protein precipitated by addition of 200 µL CH3OH followed by 600 µL CH3CN. The mixtures were centrifuged and the supernatants were filtered (UF-filter AcroPrepTM Advance 96 Filter plates 30K, Pall Corporation) before being diluted with H2O to 10 % organic solvent. Samples were injected (10 µL, Full loop injection overfill factor 4) onto an ACQUITY UPLC CSH Fluoro Phenyl Column (1.7 µm, 2.1 x 100 mm, Waters). Separation was achieved using a binary gradient at a flow rate of 0.4 mL/min. Mobile phase A was 0.2 % formic acid in H2O and mobile phase B was 0.1 % formic acid in MeOH. Milli-Q H2O and LC-MS grade MeOH (Sigma-Aldrich) were used for eluents. Adduct 13a were separated using a linear gradient, starting at 10 % mobile phase B for 0.5 min. The gradient was changed to 40 % B over 0.1 min and then to 80 % B over 2.4 min. The retention time for 13a was 2.91 min. The mass spectrometer was set to operate in the positive ion mode with multiple reaction monitoring (MRM) experiments applied. Adduct 13a specific MRM transitions were 684.4/284.2 and 684.4/238.1 [m/z] for the quantification and qualification transition respectively. All data acquisition and processing was performed using MassLynx 4.1 SCN 714 (Waters). Recombinant Antibody Development. From the single domain phage antibody library “pREDATOR”30 a single phage display selection round against adduct 13a was performed. The phage antibodies were evaluated for their ability to bind 13a by ELISA analysis. The genes encoding the best performing antibody candidates were cloned into pET22-b (+) and pMJ-LEXSY-rFc vectors using


Page 3 of 7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


restriction sites NcoI and NotI. After ligation the vectors were transformed in BL21-Gold and transfected in Leishmania tarentolae T7-TR.30,31 The single domain and

rabbit Fc fused single domain antibodies were expressed into the supernatant and then purified on Protein A HP spinTrap

Figure 2: a) Reaction between AcAcO 1a and Friedländer probes 2, 10, and 12. b) Structure of tested electrophilic probes 4, 6, 8. c) 10 µM meta- and para-Friedländer probes 2 and 10 were added to plasma spiked with 0, 10 or 50 µM AcAcO. The pH was unadjusted. Reactions were made in duplicates and allowed to react for 5 days at rt. The concentrations of 3 and 11 were quantified by UPLC-MS/MS analysis. d) Standard addition experiments in human plasma; 10 µM bifunctional Friedländer probe 12 was added to plasma spiked with 0, 50 and 200 µM AcAcO. The pH was adjusted to 9. Reactions were made in triplicates and allowed to react for 2 or 5 days at 40 °C. The concentration of 13a was quantified by UPLC-MS/MS analysis. e) 13a concentrations from the standard addition experiment plotted against spiked AcAcO levels and the data was fitted with a linear regression. The error bars represent one standard deviation. Intercept between the linear regression and the x-axis represents the (negative) endogenous concentrations of AcAcO.

(GE Healthcare). All antibodies were evaluated by ELISA for their ability to bind the bifunctional probe 12, the adduct 13a and MPBS as negative control. AML12 Hepatocyte Experiment. AML12 hepatocytes were seeded at 10000 cells/well in a black 96-well plate (Thermo-Fisher Nuncleon coated) in 100 µL complete culture medium; DMEM/F12 1:1 mixture with 1% P/S, 1% ITS solution (Insulin-Transferrin-Selenium solution) and 10% FBS (all from Thermo-Fisher). After 24h the medium was removed and replaced with 100 µL complete culture medium augmented with 2 mM sodium octanoate (Sigma-Aldrich) or 2 mM sodium octanoate and 50 µM WY-14643 (Selleckhem), or serum free medium (no FBS) containing 2 mM sodium octanoate. After 72 h, 60 µL of culture medium was transferred to a clear 96-well plate

containing 5 µL of 252 mM carbonate solution and 5 µL of 140 µM Friedländer probe 12 in each well to initiate the ReactELISA analysis. The remaining medium was discarded and 100 µL of complete culture medium was added along with 20 µL of cell titer blue (Promega). After 1 h incubation, fluorescence intensity at 584 nm was recorded (Tecan Spark 10M plate reader). The fluorescence count was used to normalize the ELISA data to the number of living cells. Adduct 13a formation was then analyzed by the described ELISA protocol.

Results and Discussion Development of Bifunctional Capture Probe. As the capture group of our bifunctional probe, we initially chose a trifluoromethyl ketone meta-Friedländer moiety 2



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

inspired by very early literature precedence on the Friedländer reaction.32 We found that this type of capture group reacts with β-carbonyl compounds, such as AcAcO, and generates a robust epitope for antibody recognition (Figure 2a). The meta-Friedländer probe 2 was compared to a panel of simpler electrophilic probes e.g. phenyl triazolidinone (PTAD) 4, a phenyl diazonium salt 6 and a phenyl aldehyde 8 (Figure 2b), for its potential to undergo a chemoselective reaction with AcAcO in either buffer or plasma sample containing hundreds of other N-, O- and S-nucleophilic endogenous species.24,25,33-35 Encouragingly, the meta-Friedländer probe 2 displayed high reactivity towards AcAcO in plasma and furthermore showed a directly proportional increase in product formation (Figure S1). These results were superior to those obtained with the simpler electrophilic probes. These findings indicate a high tolerance to other endogenous nitrogen and thiol nucleophiles. Based on these results we prepared the equivalent paraFriedländer probe 10, which we found more reactive than its meta counterpart 2 (Figure 2a,c). Product formation increased for up to 5 days, which is a feasible reaction time for further application of the reaction (Figure S2). Finally, we synthesized a complete bifunctional paraFriedländer probe 12 to test whether the biotinylated linker was detrimental to the reactivity. The results however convincingly demonstrated that also 12 dosedependently and robustly reflected levels of AcAcO in a plasma sample (Figure 2d and Figure S3a). The amount of probe adduct 13a correlated directly with AcAcO and the endogenous concentration of AcAcO could be estimated to ~50 µM – a normal endogenous plasma level - by simple extrapolation of the data i.e. at 5 days conversion at rt and 2 and 5 days conversion at 40 °C (Figure 2e and Figure S3b).36 The data from 2 days at rt extrapolated slightly higher to around 70 µM. A finding that indicated that higher conversion likely will be beneficial for method accuracy. The chemoselectivity of the new probe was finally validated in test reactions with endogenous nucleophiles known to interfere or react with established assays or reagents.25,35 We found that neither tyrosine, tryptophan, nor cysteine that otherwise reacts smoothly with aromatic aldehydes,35 reacted with 12, however the β-ketoacid oxaloacetate 1b generated the adduct 13b (Figure 3). Other β-carbonyl compounds, potentially present in biological samples: 2-methylacetoacetate, 3-oxohexanoic acid, and malondialdehyde, did not affect levels of the acetoacetate adduct 13a generated as determined by LCMS/MS or the final ELISA readout in competition experiments (Figure S4). Though we have tested interference from well-known carbon nucleophilic metabolites, the possibility that interference may occur from other presently unknown carbon nucleophilic metabolites or exogenous compounds/drugs that can undergo addition to the Friedländer probe exist.

Page 4 of 7

phage antibodies can be directly selected against immobilized 13a in streptavidin-coated wells (Figure S8).37 For our screening we used the in-house “pREDATOR” single domain antibody library.30 The nonbinding phage antibodies were washed away, and the bound phage displaying antibodies with affinity towards 13a were eluted and infected into E. Coli and plated. Twenty-one monoclonal colonies produced phage antibodies that were able to bind to 13a (Figure S9) with six clones having high specificity for 13a (Figure S10 and S11). To produce soluble single domain antibody fragments, the genes encoding the six antibody fragments were sub-cloned into a pET22b(+) vector and expressed in E. Coli. Five of the six candidates produced soluble single domain antibodies and two maintained their specificity towards 13a (Figure S12). Finally, to increase stability and affinity through the avidity effect the two antibody fragments were expressed in Leishmania tarentolae as dimers of rabbit Fc fusion proteins.31 Of the two Fc fusion antibodies Ab1 kept its specificity towards 13a (Figure S13). Furthermore, it was found that Ab1 displayed no cross-reactivity towards the probe 12 or the oxaloacetate adduct 13b (Figure 3a), the antibody also did not display any cross reactivity towards the malondialdehyde adduct mixture (Figure S4a,b). To further test the selectivity of the antibody, adduct mixtures of structurally very similar compounds (2-methylacetoacetate and 3-oxohexanoic acid) were also examined. As expected some cross reactivity was observed towards these mixtures (Figure S4a,b). However, these compounds have not been quantified under disease free conditions, according to the Human Metabolome Database,38 nor did they interfere with the assay (Figure S4c,d). The new antibody Ab1 was tested in a series of calibration samples prepared from plasma spiked with 10 µM Friedländer probe 12 and increasing levels of AcAcO (02000 µM). From the ELISA calibration data (Figure 3c) it is clear that the recombinant antibody Ab1 performs well and the estimated dynamic range of the assay is around 60-1000 µM AcAcO which is suitable for blood AcAcO measurements.

Recombinant Antibody Selection and Testing. For selection of a specific recombinant antibody, phage display techniques is particularly well suited, as binding


Page 5 of 7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. a) Specificity assay of Ab1. ELISA readout from wells coated with 12, 13a, 13b or MPBS using Ab1 as primary antibody in varying concentrations. b) Chemical structure of 12, 13a, and 13b. c) Friedländer ReactELISA. 10 µM Friedländer probe 12 was added to plasma spiked with AcAcO 0-2000 µM and the pH was adjusted to 9.0. The samples were then allowed to react for 5 days at 40 °C. AcAcO levels (endogenous + spiked) were plotted against ELISA readout and fitted using a four parameter logistic (4PL) regression. All reactions were performed in triplicate. d) Recovery of AcAcO determined by ELISA. The error bars represent one standard deviation. To verify, we spiked two unrelated plasma samples with varying amounts of AcAcO (100, 200, and 500 µM). The determined recovery varied between 86-122 % (Figure 3d, see also Figure S15 and Table S1).

Cell Based Experiments. Having established a robust procedure for determining AcAcO in plasma, we focused our attention towards adapting and optimizing the assay for 96-well format to enable AcAcO measurements in culture medium from liver cells. Apart from miniaturizing the format, anticipated levels of AcAcO in culture media are also lower than in plasma.12 We therefore fine-tuned two key assay parameters; reaction pH and TMB substrate incubation time (data not shown), and ended at a setup with pH 9.75 and 20 min TMB substrate incubation time that with statistically significance could detect 5 µM AcAcO spiked to culture media (Figure S6). The endogenous level of AcAcO in the fresh culture media as determined by HS-GC-FID was 10 µM, explaining the signal in the culture media blank. Curiously, during the final studies we also discovered that even though the aldol reaction proceeds smoothly in phosphate buffer (Figure S1) the related Friedländer reaction progress slowly in the same media (Figure S5). To test whether this was due to amino acid organocatalysis endogenous levels of proline and lysine were added to the reactions, however only slightly increasing reaction conversion as determined by LC-MS/MS (Figure S5).34 Which serum or growth media factor(-s) that increases reaction rate is therefore presently unknown. As the reactions in growth media however gave a satisfactory conversion we moved on with the optimized assay conditions to pursue cell based experiments. Treatment of AML12 hepatocytes with known modulators of ketogenesis in the presence of sodium octanoate (NaOct) produced ketogenic cell populations (Figure 4a).12 The ReactELISA assay was reliably capable of detecting minor perturbations to the system. Addition of 50 µM WY-14643, a PPARα agonist with anti-aging effects,39 to culture media produced a 3-fold increase in relative signal. This demonstrated an increased production of AcAcO in response to PPARα stimulation. Culturing under serum-free conditions without insulin (ITS solution omitted from media), produced a 9-fold increase in relative signal, demonstrating the key regulatory role of insulin in balancing glucose and lipid metabolism.40

Figure 4. a) Perturbations that affect the ketogenic state of AML12 cells. b) Detection of AcAcO from AML12 culture media after incubation for 72h with medium containing NaOct (2 mM), NaOct (2 mM) and WY-14643 (50 µM), or NaOct (2 mM) in serum-free medium without insulin. Results are representative for four individual experiments, each performed in triplicates. ** p < 0.01 (t-test). c) Structure of NaOct and WY-14643. The error bars represent one standard deviation.

Conclusions In conclusion we have demonstrated that Friedländer substrates react with a high degree of chemoselectivity with endogenous AcAcO in complex biological matrices such as plasma or cell culture media. The Friedländer substrates are superior to several previously applied Creactive electrophiles such as azodicarboxylates, PTAD, diazonium salts, and aldehydes, in regard to chemoselectivity, stability, and dynamics and we envision that the Friedländer reaction will find broader use in chemical biology. On the basis of this chemoselective reaction, we developed a conceptually novel, robust and operationally simple ReactELISA method for assaying the carbon nucleophilic metabolite AcAcO in human plasma and in liver cell cultures. Due to both the Friedländer reaction and the high specificity of the recombinant antibody developed, the assay has several decisive advantages compared to the earlier known colorimetric or indirect enzymatic methods for monitoring AcAcO. In order to get sufficient conversion in the Friedländer capture reaction, the assay needs to incubate 3-5 days before the ELISA procedure can commence. As AcAcO is relatively stable under the assay conditions (Figure S7c) and samples are prepared and stabilized by simple addition of 12 to microplates with the culture media, the assay incubation time poses no greater obstacle for application of the assay as a new screening tool for small molecules or other interventions that increases lipid metabolism - a key indicator for healthy aging. The assay was tested for interference from known endogenous carbon nucleophilic metabolites, but as the assay relies on a Friedländer reaction, carbon nucleophilic drugs or other exogenous compounds discovered e.g. in a high-throughput



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

screen or biological experiment should most adequately be tested for interference with the assay in control experiments.

The assay in the current format was able to detect levels of AcAcO from around 5 µM to high µM levels, a range that encompasses many naturally occurring metabolites. We however hope that the ReactELISA concept will find broader use for quantitation of other important metabolites and in particular for reactive, unstable and very low (< 1 µM) level compounds where robust and simple methods are scarce.41,42 Such studies are currently underway in our laboratories and will be reported in due course.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:XXXXXX. Supplementary figures and tables. Experimental methods including synthetic procedures. Characterization data including antibody sequence (PDF).

AUTHOR INFORMATION Corresponding Author [email protected]

ACKNOWLEDGMENT Mette H. Mantel is acknowledged for doing preliminary experiments. MJ thanks the Danish Research Council for Independent Research, Medical Sciences and the Lundbeck Foundation (R180-2014-2740) and TBP the Lundbeck Foundation (Fellow grant R105-A9308) for financial support.

REFERENCES (1) Balasse, E. O.; Féry, F. Diabetes Metab. Rev. 1989, 5, 247–270. (2) Siess, E. A.; Kientsch-Engel, R. I.; Wieland, O. H. Eur. J. Biochem. 1982, 121, 493. (3) Colman, R. J.; Beasley, T. M.; Kemnitz, J. W.; Johnson, S. C.; Weindruch, R.; Anderson, R. M. Nat Commun. 2014, 5, 3557. (4) Koubova, J.; Guarente, L. Genes Dev. 2003, 17, 313–321. (5) Shimazu, T.; Hirschey, M. D.; Newman, J.; He, W.; Shirakawa, K.; Moan, N. L.; Grueter, C. A.; Lim, H.; Saunders, L. R.; Stevens, R. D.; Newgard, C. B.; Farese, R. V.; Cabo, R. de; Ulrich, S.; Akassoglou, K.; Verdin, E. Science 2013, 339, 211–214. (6) Veech, R. L. Prostaglandins, Leukot. Essent. Fatty Acids 2004, 70, 309–319. (7) Wang, M. C.; O’Rourke, E. J.; Ruvkun, G. Science 2008, 322, 957–960. (8) Xie, T. Science 2008, 322, 865–866. (9) Newman, J. C.; Verdin, E. Trends Endocrinol. Metab. 2014, 25, 42–52. (10) Wood, J. G.; Rogina, B.; Lavu, S.; Howitz, K.; Helfand, S. L.; Tatar, M.; Sinclair, D. Nature 2004, 430, 686–689. (11) Harrison, D. E.; Strong, R.; Sharp, Z. D.; Nelson, J. F.; Astle, C. M.; Flurkey, K.; Nadon, N. L.; Wilkinson, J. E.; Frenkel, K.; Carter, C. S.; Pahor, M.; Javors, M. A.; Fernandez, E.; Miller, R. A. Nature 2009, 460, 392–395. (12) Sengupta, S.; Peterson, T. R.; Laplante, M.; Oh, S.; Sabatini, D. M. Nature 2010, 468, 1100–1104. (13) Ajmo, J. M.; Liang, X.; Rogers, C. Q.; Pennock, B.; You, M. Am. J. Physiol. Gastrointest. Liver Physiol. 2008, 295, G833–G842. (14) Gano, L. B.; Patel, M.; Rho, J. M. J. Lipid Res. 2014, 55, 2211– 2228.

Page 6 of 7

(15) Zou, X.; Meng, J.; Li, L.; Han, W.; Li, C.; Zhong, R.; Miao, X.; Cai, J.; Zhang, Y.; Zhu, D. J. Biol. Chem. 2016, 291, 2181–2195. (16) Woolf, E. C.; Scheck, A. C. J. Lipid Res. 2015, 56, 5–10. (17) Tran, M. T.; Zsengeller, Z. K.; Berg, A. H.; Khankin, E. V.; Bhasin, M. K.; Kim, W.; Clish, C. B.; Stillman, I. E.; Karumanchi, S. A.; Rhee, E. P.; Parikh, S. M. Nature 2016, 531, 528–532. (18) Massieu, L.; Haces, M. L.; Montiel, T.; Hernández-Fonseca, K. Neurosci. 2003, 120, 365–378. (19) Fine, E. J.; Miller, A.; Quadros, E. V.; Sequeira, J. M.; Feinman, R. D. Cancer Cell Int. 2009, 9, 14. (20) Kang, H.-B.; Fan, J.; Lin, R.; Elf, S.; Ji, Q.; Zhao, L.; Jin, L.; Seo, J. H.; Shan, C.; Arbiser, J. L.; Cohen, C.; Brat, D.; Miziorko, H. M.; Kim, E.; Abdel-Wahab, O.; Merghoub, T.; Fröhling, S.; Scholl, C.; Tamayo, P.; Barbie, D. A.; Zhou, L.; Pollack, B. P.; Fisher, K.; Kudchadkar, R. R.; Lawson, D. H.; Sica, G.; Rossi, M.; Lonial, S.; Khoury, H. J.; Khuri, F. R.; Lee, B. H.; Boggon, T. J.; He, C.; Kang, S.; Chen, J. Molecular Cell 2015, 59, 345–358. (21) Fan, M.; He, J. In Trends in Immunolabelled and Related Techniques, InTech, Rijeka, 2012; pp 53–66. (22) Lim, S. L.; Ichinose, H.; Shinoda, T.; Ueda, H. Anal. Chem. 2007, 79, 6193–6200. (23) Ma, S.; Subramanian, R. J. Mass Spectrom. 2006, 41, 1121–1139. (24) Harano, Y.; Kosugi, K.; Hyosu, T.; Uno, S.; Ichikawa, Y.; Shigeta, Y. Clin. Chim. Acta. 1983, 134, 327–336. (25) Yamato, S.; Kobayashi, K.; Ebara, K.; Shimada, K.; Ohta, S. Biol. Pharm. Bull. 2003, 26, 397–400. (26) Williamson, D. H.; Mellanby, J.; Krebs, H. A. Biochem. J. 1962, 82, 90–96. (27) Yamato, S.; Shinohara, K.; Nakagawa, S.; Kubota, A.; Inamura, K.; Watanabe, G.; Hirayama, S.; Miida, T.; Ohta, S. Anal. Biochem. 2009, 384, 145–150. (28) Créminon, C.; Taran, F. Chem. Commun. 2015, 51, 7996– 8009. (29) Tawfik, D. S.; Green, B. S.; Chap, R.; Sela, M.; Eshhar, Z. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 373–377. (30) Mandrup, O. A.; Friis, N. A.; Lykkemark, S.; Just, J.; Kristensen, P. PLOS ONE 2013, 8, e76834. (31) Jørgensen, M. L.; Friis, N. A.; Just, J.; Madsen, P.; Petersen, S. V.; Kristensen, P. Microb. Cell Fact. 2014, 13, 9. (32) Schöpf, C.; Lehmann, G. Justus Liebigs Ann. Chem. 1932, 497, 7–21. (33) Ban, H.; Gavrilyuk, J.; Barbas, C. F. J. Am. Chem. Soc. 2010, 132, 1523–1525. (34) Alberg, D. G.; Poulsen, T. B.; Bertelsen, S.; Christensen, K. L.; Birkler, R. D.; Johannsen, M.; Jørgensen, K. A. Bioorg. Med. Chem. Lett. 2009, 19, 3888–3891. (35) Tanaka, F.; Mase, N.; Barbas, C. F. Chem. Commun. 2004, 15, 1762–1763. (36) Harris, D. C. Quantitative Chemical Analysis 6th Edition; W.H. Freeman: New York, 2003 (37) McCafferty, J.; Schofield, D. Curr. Opin. Chem. Biol. 2015, 26, 16–24. (38) Wishart, W. S.; Jewison, T.; Guo, A. C.; Wilson, M.; Knox, C. et al., Nucleic Acids Res., 2013, 41, D801-D807 (39) Poynter, M. E.; Daynes, R. A. J. Biol. Chem. 1998, 273, 32833– 32841. (40) Saltiel, A. R.; Kahn, C. R. Nature 2001, 414, 799–806. (41) Dhar, A.; Desai, K.; Liu, J.; Wu, L. J. Chromatogr. B. 2009, 877, 1093–1100. (42) Esterbauer, H.; Zollner, H. Free Radic. Biol. Med. 1989, 7, 197–203.


Page 7 of 7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Insert Table of Contents artwork here


ReactELISA: Monitoring a Carbon Nucleophilic ... - ACS Publications

Recommend Documents