Alteraciones en las acilcarnitinas detectadas por MS/MS

2.17.1 Fluorescence spectra

All fluorescence spectra were acquired using a single sample unit PerkinElmer LS 55 luminescence spectrometer (5 nm slit width, 1 nm interval, 1 second integration) and an excitation wavelength of 435 nm. Fluorescence emission spectra were recorded from 400 to 600 nm. Samples were measured in cuvettes (3.5 mL volume, 10 mm path length, Sarstedt). The samples were diluted in FRET buffer (20 mM Tris-HCl pH 7.4 buffer containing, 0.5 mM DTT, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA and 5% v/v glycerol) to a final volume of 3 mL. The cuvettes were sealed with parafilm and inverted 10 times. Fluorescence emission spectra of the FRET pair and eCFP-TEV-Nrf2 and eYFP-TEV-Kelch were recorded separately. All samples were measured in duplicate.

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2.17.2 FRET efficiency and sensitised emission

The observed decrease in donor emission at 475 nm of the donor and acceptor pair relative to that of the donor alone was used to calculate the efficiency of energy transfer (FRET efficiency or FE) between the fluorescent fusion proteins:

FE = 1 −F

da

Fd

Where da = donor emission in the presence of the acceptor and d = donor emission in the absence of the acceptor.

The observed increase in acceptor emission at 527 nm of the donor and acceptor pair relative to that of the acceptor alone was used to calculate the sensitised emission (SE) of the acceptor fluorescence:

SE = F

ad

Fa − 1 *

(εa * ca) (εd * cd)

Where ad = acceptor emission in the presence of the donor, a = acceptor emission in the absence of the donor, εa or εd = extinction coefficient of the acceptor or donor respectively and ca or cd = molar concentration of the acceptor or donor respectively.

2.17.3 FRET eCFP-TEV-eYFP validation: ProTEV protease

A series of 100 µL reactions were prepared in duplicate containing increasing amounts of ProTEV protease (Promega) (0.5, 1.0, 1.5, 2.0, 2.5, 5.0 and 10 units). Each reaction contained 20 µg purified eCFP-TEV-eYFP protein, 1x ProTEV buffer and 1 mM DTT, which was made to a total of 100 µL with dH2O. The mixture was vortexed and pelleted at 13,000 x g for 10 sec. A control reaction containing the fusion protein but no ProTEV protease was prepared in parallel. Both test and control reactions were incubated at 30 °C for 30 min. After incubation, 20 µL aliquots of the reaction mixtures were removed for SDS-PAGE analysis (2.14) and stored at - 20 °C. The remaining 80 µL reaction mixtures were transferred to cuvettes containing FRET buffer.

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2.17.4 FRET pair: eCFP-TEV-Nrf2 and eYFP-TEV-Kelch titration

Following the determination of the eCFP-TEV-Nrf2 and eYFP-TEV-Kelch protein concentrations, the protein mixtures were diluted as appropriate in FRET buffer. Final concentrations of 0.05, 0.07, 0.09, 0.11, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70 and 0.80 µM eYFP-TEV-Kelch or unconjugated eYFP were added to a final concentration of 0.11 µM eCFP-TEV-Nrf2. Protein samples containing either the donor or acceptor were prepared separately.

Emission spectra were recorded and FRET efficiencies and sensitised emissions were calculated for each protein combination. Binding curves were fitted by nonlinear regression using SigmaPlot software (ligand binding, one site saturation) and Kd and Bmax values were determined.

In addition, the emission intensity values at 527 nm were plotted for eCFP-TEV-Nrf2 titrated with unconjugated eYFP and for direct excitation of eYFP-TEV-Kelch. Linear regression was used to connect the data points and generate slope values:

y = ax+b

Where y represents the emission at 527 nm, a is the extent of emission increase upon titration of eCFP-TEV-Nrf2 with increasing concentration of unconjugated eYFP or as a result of the direct excitation of eYFP-TEV-Kelch and b is the emission at 527 nm when the concentration of unconjugated eYFP or eYFP-TEV-Kelch (x) is 0 µM (y intercept).

2.17.5 FRET buffer system optimisation: NaCl

A series of solutions were made containing increasing amounts of NaCl (0, 50, 100 and 150 mM) in the original FRET buffer (without NaCl where appropriate). Each mixture contained 0.11 µM eCFP-TEV-Nrf2 and 0.30 µM eYFP-TEV-Kelch. Fluorescence emission spectra were recorded.

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2.17.6 FRET pair: eCFP-TEV-Nrf2 and eYFP-TEV-Kelch validation: ProTEV protease

A series of 100 µL reactions were prepared containing increasing amounts of ProTEV protease (2.5, 5.0, 7.5 and 10 units). Each reaction contained 8.9 µg eCFP- TEV-Nr2 and 24.3 µg eYFP-TEV-Kelch, 1x ProTEV buffer, 1 mM DTT and dH2O. The mixture was vortexed and pelleted at 13,000 x g for 10 sec. A control reaction containing the fusion proteins but no ProTEV protease was prepared in parallel. The reactions were incubated at 30°C for 30 min. Following incubation, 20 µL aliquots of the reaction mixtures were removed for SDS-PAGE analysis (Section 2.14) and stored at - 20°C. The remaining 80 µL reaction mixtures were transferred to cuvettes containing FRET buffer. Fluorescence emission spectra were recorded.

2.17.7 Inhibition of FRET between FRET pair: eCFP-TEV-Nrf2 and eYFP-TEV- Kelch

Vehicle (DMSO) concentration

A series of solutions in FRET buffer were made containing increasing percentages of DMSO (0, 1, 5 and 10%). Each mixture contained 0.11 µM eCFP-TEV-Nrf2 and 0.30 µM eYFP-TEV-Kelch. Fluorescence emission spectra were recorded.

Nrf2 peptide inhibitor

Solutions in FRET buffer were made containing 0.11 µM eCFP-TEV-Nrf2 and 0.30 µM eYFP-TEV-Kelch in the presence or absence of 10 µM of an unlabelled Nrf2 derived peptide inhibitor (in 0.1% DMSO). Fluorescence emission spectra were recorded.

2.17.8 FRET - multi-well plate format

All fluorescence spectra were acquired using a Pherastar BMG Labtech microplate reader (excitation filter: 430 nm, dual emission filters: 480 nm and 530 nm). The

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samples were diluted in FRET buffer (20 mM Tris-HCl pH 7.4 buffer containing, 0.5 mM DTT, 0.1 mM EDTA and 5% v/v glycerol) to a final volume of 100 µL.The untreated black 96-well microtiter plates (Corning) were covered with aluminium foil to protect the fluorophore-fused proteins from light and incubated at 800 rpm on a plate shaker for 4 min. Fluorescence emission spectra of the FRET pair and eCFP- TEV-Nrf2 and eYFP-TEV-Kelch were recorded separately. All samples were measured in triplicate.

2.17.9 FRET titration - multi-well plate format

Following the determination of the eCFP-TEV-Nrf2 and eYFP-TEV-Kelch protein concentrations, the protein mixtures were diluted as appropriate in FRET buffer. Final concentrations of 0.01, 0.03, 0.05, 0.07, 0.09, 0.11, 0.20, 0.30, 0.40 and 0.50 µM eYFP-TEV-Kelch were added to a final concentration of 0.11 µM

eCFP-TEV-Nrf2 or eCFP-TEV-Nrf2 mutant proteins. Emission intensity values at 480 nm and 530 nm were recorded and FRET efficiencies were calculated for each protein combination. Binding curves were fitted by nonlinear regression using SigmaPlot software (ligand binding, one site saturation) and Kd and Bmax values were determined.

2.17.10 FRET competition – multi-well plate format

In this multi-well plate format concentrations of 0.11 µM eCFP-TEV-Nrf2 and 0.20 µM eYFP-TEV-Kelch were used (~ 80% of maximal FE). Assays were performed with increasing concentrations of peptide inhibitor (0.001 – 100 µM) at a final volume of 100 µL and a final DMSO concentration of 0.1% v/v. All measurements were carried out in triplicate. Plates were read directly after mixing the components. The percentage inhibition was determined using:

% FE = 1 −FE

vehicle

− FEinhibitor

FEvehicle *100

Inhibition curves were fitted to a standard four-parameter logistic function using SigmaPlot and IC50 values were determined.

63 2.17.11 Calculation of Z′ value

The suitability of this assay for high throughput screening (HTS) was assessed by determining the Z′ value. This parameter is used as a measure of assay performance, based on the maximal and minimal fluorescence intensity at 480 nm. The Z′ value was calculated using the equation:

Z' = 1 − 3SDmaxFRET− 3SDminFRET maxFRET480nm− minFRET480nm

Where min FRET480nm = the minimal fluorescence intensity observed in the absence of any inhibitor, max FRET480nm = the maximal fluorescence intensity observed in the presence of an inhibitor, and SD = the standard deviation of the fluorescence emissions at 480 nm. For both minimal and maximal fluorescence signal 0.11 µM eCFP-TEV-Nrf2 and 0.20 µM eYFP-TEV-Kelch was pipetted into the wells. The min FRET480nm condition was obtained using a final concentration of 0.1% v/v DMSO and the max FRET480nm condition was acquired using 100 µM of the 7-mer stearic acid (St) conjugated peptide St-DPETGEL in a final concentration of 0.1% v/v DMSO. The fluorescence emission at 480 nm of both the minimal and maximal fluorescence signal was measured in 48 wells of a 96-well plate. Triplicate experiments were performed.