Anis Elloumi

Lindsay Mas-Normand

Jamie Bride

Guillaume Reversat

Valérie Bultel-Poncé

Alexandre Guy

Camille Oger

Marie Demion

Jean-Yves Le Guennec

Thierry Durand

Claire Vigor

Ángel Sánchez-Illana

Jean-Marie Galano
email: jean-marie.galano@umontpellier.fr.

From MS/MS library implementation to molecular networks: Exploring oxylipin diversity with NEO-MSMS

Oxylipins, small polar molecules derived from the peroxidation of polyunsaturated fatty acids (PUFAs), serve as biomarkers for many diseases and play crucial roles in human physiology and inflammation. Despite their significance, many non-enzymatic oxygenated metabolites of PUFAs (NEO-PUFAs) remain poorly reported, resulting in a lack of public datasets of experimental data and limiting their dereplication in further studies. To overcome this limitation, we constructed a high-resolution tandem mass spectrometry (MS/MS) dataset comprising pure NEO-PUFAs (both commercial and self-synthesized) and in vitro free radical-induced oxidation of diverse PUFAs. By employing molecular networking techniques with this dataset and the existent ones in public repositories, we successfully mapped a wide range of NEO-PUFAs, expanding the strategies for annotating oxylipins, and NEO-PUFAs and offering a novel workflow for profiling these molecules in biological samples.

Background & Summary

Lipid peroxidation was mechanistically revealed in the 1940s thanks to the scientists at the British Rubber Products Association, and only then, the role of free radicals derived from molecular oxygen started to make sense leading to the understanding of the autooxidation phenomena1. Soon enough, the discovery of arachidonic acid (AA) cascade (enzymecatalyzed peroxidation) led to the discovery of bioactive eicosanoids, and oxylipins produced by other polyunsaturated fatty acids (PUFA) than AA2. Finally, non-enzymatic oxygenated metabolites of polyunsaturated fatty acids (NEO-PUFAs) generated by triplet or singlet oxygen-dependent reactions were originally identified as mere product of oxidative stress (hence potential biomarkers), however several lines of evidence have highlighted their role as lipid mediators such as the isoprostanoids3.

Early methods for detection and quantification of oxylipins and NEO-PUFAs in samples relied on thin-layer chromatography (TLC), gas chromatography (GC), and mass spectrometry (MS). Over the past three decades, advances in hyphenated techniques such as liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) along with high resolution MS and novel data analysis approaches have allowed the comprehensive analysis of the various modifications of lipids by enzymatic and non-enzymatic reactions, forming higher levels of structural complexity, known as the epilipidome4.

One of the challenges in epilipidomics is the identification of the modified lipids, especially so if they were already discovered, i.e., the dereplication of oxidized lipids5.

While efforts are being currently underway for the practical identification of oxylipins esterified to complex lipids6, a recent work by Watrous et al.7 reported the dereplication of free oxylipins by molecular networking8, 9 using a high resolution mass spectrometry (HRMS) directed non-targeted approach, thanks to reference tandem mass spectra of 234 of commercially available oxylipins, and a few NEO-PUFAs. Interestingly, they reported the discovery of more than 500 novel oxylipins (called "putative" in the text). This number should have raised more interest to the community, as it is believed that very little gap remains for novel oxylipin structures. We initially hypothesized that the lack of MS/MS libraries for NEO-PUFAs could partially explain the "putative" oxylipins number. Although a large amount of knowledge over the last 25 years has been accumulated concerning the relevant NEO-PUFAs detected by LC-MS/MS, it appears impossible to gather full spectra MS/MS data from either the reports of their discovery or from their synthetic preparation. Knowing that targeted MRM (multiple reaction monitoring) on low resolution mass spectrometry is preferred for quantification of NEO-PUFAs, full MS/MS data repositories could seem irrelevant at the time of their discovery. However very few of those discovered NEO-PUFAs were made available making todays dereplication not feasible. This problematic is recurrent in the field, and a recent investigation proved the importance of data repositories10. Recent studies toward the development and optimization of analytical strategies based on HRMS showed great promises, and improvement of spectrometer sensitivity will surely keep on improving11,12 allowing mainstream use.

As part of our continuing interest in NEO-PUFA chemistry, we developed a streamlined molecular networking dereplication pipeline based on the implementation of a MS/MS library that we named non-enzymatic oxylipins MS/MS library (NEO-MSMS). It contents an in house collections of synthesized and unique NEO-PUFA standards, commercially available enzymatic oxylipins, and a curated MS/MS data collection of commercially available oxylipins from Watrous et al. study7. Moreover, in vitro non-enzymatic oxidation of the four main PUFA (AA, eicosapentaenoic acid [EPA], docosahexaenoic acid [DHA] and α Linolenic acid [ALA]) lead to the creation of a vast MS/MS dataset of NEO-PUFAs which was sought to extend the coverage signature of oxylipins. This data descriptor presents the deposition of all this data in Zenodo and MASSIVE repositories along with its subsequent technical validation. Additionally, we also present the inclusion of the NEO-MSMS as a compatible GNPS library augmented with putatively annotated MS/MS oxylipin spectra extracted from the in vitro oxidation (Fig. 1).

We believe this information could set the ground for understanding the importance of building a MS/MS library dedicated to oxygenated PUFA metabolites, and we hope natural product researchers, organic chemists and lipidomists in the field to share their collections, expand and use NEO-MSMS.

Methods

Standards and reagents.

All solvents and reagents employed were ≥98% purity unless otherwise indicated, including cyclohexane, acetonitrile, methanol, ethanol absolute, ammonia solution 30% (w/w), and water, all sourced from Fisher Chemicals (Waltham, MA, USA). Hexane, ethyl acetate, potassium hydroxide, sodium hydroxide, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, monosodium phosphate, disodium phosphate, glucose, trimethyl phosphite, acetic acid, and formic acid were obtained from Merck (Darmstadt, Germany). The free radical initiator 2, 2′-azobis(4-methoxy-2, 4-dimethylvaleronitrile) (V70) was procured from FUJIFILM Wako Chemicals (Tokyo, Japan) and N-methyl benzohydroxamic acid (NMBHA) was prepared in our laboratory following the methodology outlined in a prior publication13. Tyrode's solution was prepared weighting and dissolving in water the appropriated amounts of the following salts to obtain the concentration of NaCl 8 g/L, KCl 0.2 g/L, CaCl2 0.2 g/L, MgCl2 0.1 g/L, NaH2PO4 0.05 g/L, NaHCO3 1 g/L, and glucose 1 g/L. The pH was adjusted with 1 M solution NaOH to 7.4.

The PUFAs used in the in vitro oxidation were AA and EPA from Merck (Darmstadt, Germany), DHA from Santa Cruz Biotechnology (Dallas, TX, USA), and ALA from BLDpharma (Shanghai, China). The PUFA esters employed were DHA ethyl ester, AA methyl ester, and EPA methyl ester from Merck (Darmstadt, Germany) and ALA methyl ester purchased from Fisher Chemicals (Waltham, MA, USA).

Regarding oxylipin standards, individual stock solutions at 1 μmol/L of 131 oxylipins (purity >95%) were prepared dissolving the appropriate amounts in H2O:CH3CN mixture (i.e. 20% CH3CN). This includes 72 commercially available enzymatic oxylipins and NEO-PUFAs purchased at Cayman Chemical (Ann Arbor, MI) as individual standard or premixed solution and 59 NEO-PUFAs obtained by total synthesis in our laboratory following previously developed procedures and purity-checked by NMR14-21.

As internal standards (IS); the same mixture of oxylipins commonly employed in our laboratory for quantitative methods published elsewhere22,23 weighting the appropriate amounts of compounds and dissolving it in H2O (20% CH3CN) to obtain a concentration of 3.25 mg/L each. This includes non-natural odd number analogues of oxylipins not produced by mammal cellular process C19-16-F1t-PhytoP and C21-15-F2t-IsoP, and an isotopically labelled D4-10-epi-10-F4t-NeuroP.

In vitro radical-induced oxidation of PUFAs.

To encompass the broad spectrum of NEO-PUFAs produced via triplet oxygen-dependent reactions from PUFAs, various oxidation protocols were applied to DHA, ALA, AA, and EPA acids and esters. The objective was to generate precise samples for encompassing the chemical signature of NEO-PUFAs (oxPUFA data in the text).

Oxidation of PUFAs with H 2 O 2 .

Approximately 5 mg of free fatty acid (≈17 μmol) were dissolved in 100 μL of ethanol. Subsequently, it was mixed with a 1.8 mL Tyrode's solution and 100 μL H 2 O 2 solution (120 mM in water) to reach a final volume of 2 mL and approximate concentrations of 8.5 mM and 6 mM for PUFA and H 2 O 2 , respectively (i.e., 1.4 PUFA/H 2 O 2 molar ratio). The oxidation proceeded for 30 minutes, then the reaction mixture was divided in two (1 mL each) aliquots. In one aliquot, 0.9 mL of 1 M KOH in water was added and incubated for 30 minutes at 40 °C with mid rotation in tube rotator. After this KOH saponification, 100 μL of formic acid was added to obtain an acidic pH of 4. In the other aliquot, only 1 mL of acidified water with formic acid (pH = 4) was added.

Of note: this alkaline hydrolysis step was included as a control experiment since best practice for NEO-PUFAs profiling requires such step in the extraction procedure of plasma/tissues (to the opposite of oxylipins), and that it was reported to transform or degrade a few NEO-PUFA/oxylipin structures24,25. Other oxylipin metabolites possessing 3, 5-disubstituted 1, 2dioxolane units (endoperoxides) are also known to rearrange under basic conditions via the Kornblum-DeLaMare reaction onto ketohydroxyl derivatives (i.e; PGG 2 into PGE 2 ), however the fate of endoperoxide metabolites (derived from initial 5-exo-trig cyclization followed by oxygenation of peroxyl radical precursor) remains uncertain. On model studies26, 27, linear 1, 2-dioxolanes do not rearrange under our alkaline conditions (see Supplementary Information Figure S1). Other endoperoxide derivatives (like PGGs) were not investigated.

Each aliquot (i.e., saponified and non-saponified) was purified by automated solid phase extraction (SPE) process employing Extrahera system from Biotage (Uppsala, Sweden) and Oasis Max cartridge as stationary phase (60 mg, 30 μm particle size) from Waters (Mildford, MA, USA). The SPE procedure was optimized for the extraction of oxylipins in our previous works; 22, 23, 28 briefly, after the conditioning and sample loading, the cartridge was washed with 2 mL each of 2% NH3 solution, MeOH/formic acid (20 mM, 30:70), pure hexane, and hexane/ethyl acetate (70:30). Subsequently, elution is carried out using 1.5 mL of hexane/EtOH/acetic acid (70:29.4:0.6). Finally, each aliquot elution was divided in two again (750 μL each) and 50 μL of P(OMe) 3 was added in one of the aliquots and incubated 30 min at room temperature in order to reduce hydroperoxide derivatives and bicyclic endoperoxides, keeping intact linear endoperoxides (1, 2-dioxolanes)29. The robustness of the linear endoperoxides was demonstrated through reduction tests on an isolated compound (Supplementary Information Figure S1).

The four final samples of oxidized PUFA (oxPUFA) per every single PUFA investigated (i.e., saponified/ non-saponified with/without P(OMe) 3 reduction) were evaporated with CentriVap Vacuum Concentrator (Labconco, Kansas, MO, USA) and resuspended with 970 mL of H2O (20% CH3CN) plus 30 μL of IS mixture.

Oxidation of PUFA ethyl esters with NMBHA.

Adapted from refs. 30, 31; approximately 50 mg (≈170 μmol) of esterified fatty acid was diluted in 2 mL of anhydrous CH 3 CN, mixed with 7 mg of V70 (≈23 μmol), followed by addition of 25 mg of N-methyl benzohydroxamic acid (NMBHA) (≈170 μmol), and incubated for 24 hours opened to room air (i.e. 21% O 2 ) at 37 °C in sand bath. The oxidation product was purified to eliminate NMBHA, the unreacted PUFA and other by-products with Flash-LC employing a Reveleris X2 system from Buchi (Eastern Switzerland) and a puriflash 30SI-JP/12 g 30 μm column from Interchim (Montluçon, France). Solvent was eliminated with rotavap, resuspended in 1 mL of cyclohexane with a few drops of ethyl acetate until soluble material could be injected into the Flash-LC system. The separation gradient was cyclohexane (0.1% acetic acid) (A) and ethyl acetate (0.1% acetic acid) (B) as eluents, and the flow rate was set at 30 mL/min, (25 mL per tube). The proportion of B was gradually increased from 0% to 10% over 6.2 minutes, then to 23% over 2.3 minutes, then maintained isocratic for 4.7 minutes and increased to 100% over 0.7 minutes which was held 5 minutes for a total run time of 18.9 minutes. The elution of the unreacted PUFA, oxPUFA, and the NMBHA were controlled by TLC and only the fractions of oxPUFA were finally collected. It should be noted that the amount of NMBHA employed could be very problematic if it was injected concentrated into the LC-MS system so this cleaning step is mandatory. The oxPUFA fractions were concentrated with rotavap collecting approximately 25 mg of oxPUFA of which 5 mg were diluted in 2 mL of water to reach the same concentration as in the H 2 O 2 oxidation. Then, it was proceeded to saponification, SPE, P(OMe)3 reduction and IS dilution such as in the H 2 O 2 oxidation.

Oxidation of ethyl esters of PUFAs without NMBHA.

The last oxidation was made accordingly to the previous one but without the addition of NMBHA. Additionally, during Flash-LC purification two fractions of oxPUFA were collected, i.e., the ones before and after the hypothetical elution of NMHBA. This was done to access to the oxylipins coeluting with NMBHA and lost in the NMBHA oxidation.

In vitro radical-induced oxidation or rearrangement of oxylipins.

Microscaling the previously reported leukotriene-like formation by oxidation of 15-HETE described by Rector et al. [START_REF] Rector | Identification of the Peroxidation Products of 13-Hydroxy-γ-linolenate and 15-Hydroxyarachidonate: Mechanistic Studies on the Formation of Leukotriene-like Diols[END_REF] and retrieve the partially reported information (MS/MS data) could be an answer to redeem annotation of oxylipins uncovered in past studies. Furthermore, solvent-assisted rearrangement of PGH1 and PGH2 was microscaled with the hope of acquiring the MS/MS data of the main produced metabolites, i.e., the 15-levuglandins LGE1 and LGE 2 33.

Microscaled V70 oxidation of commercially oxylipins.

A solution of 30 μL of V70 (0.15 mol/L) in CH 3 CN was added to 50 μg of 15-HETE redisposed in chromatographic glass vials with 250 μL insert with conical bottom and quickly rotated with small angle using a Bio PTR-35 360° Multi-Functional Tube Rotator from Grant Instruments, Royston, UK) at 40 °C for 24 h in an oven. The agitation conditions were the following: 100 rpm orbital, a reciprocal rotation of 11° turning angle for 6 seconds followed by a 2° vibration for 1 second. The solution is then evaporated with vacuum concentrator and diluted in 100 μL of mobile phase before LC-MS injection.

PGH rearrangement.

Commercially available standards PGH 2 (50 μg) and PGH 1 (25 μg) were placed in chromatographic glass vials with 250 μL insert with conical bottom, dissolved in 30 μL of DMSO33-35, and mechanically agitated 1 hour at 37 °C using the tube rotator with the same parameters of microscaled V70 oxidation.

Finally, 270 μL of mobile phase were added immediately before the LC-MS injection.

Blood serum and microalgae samples.

We used four samples of oxylipin extractions from human blood serum and three from microalgal biomass to demonstrate the practical application of NEO-MSMS and oxPUFA data with real samples. Serum samples had been processed for the quantitative profiling of oxylipins/NEO-PUFAs following our extraction protocol22, 23, 28 (including the KOH saponification and the SPE like the oxPUFA samples described above) and the extracts stored at -80 °C. Blood samples of healthy donors were obtained from EFS (Etablissement Français du Sang, Montpellier). All procedures in use by the EFS are defined by the law and follow institutional guidelines.

Similarly, the microalgae samples had been extracted in the framework of a previous study including the sample grinding and multiple centrifugation and purification steps36,37.

Data acquisition.

Three different methods were developed with two different LC-MS/MS platforms. On the one hand, a Vanquish UHPLC system coupled to Q-Exactive Focus orbitrap mass spectrometer from Thermo Fisher Scientific (Waltham, MA, USA) was used for the LC-MS/MS spectra acquisition of standards (method 1). On the other hand, a Vanquish UHPLC system coupled to Orbitrap ID-X Tribrid mass spectrometer, also from Thermo Fisher Scientific, was used for the Flow injection analysis-MS/MS (FIA-MS/MS) spectra acquisition of standards (method 2); and for the LC-MS/MS spectra acquisition of standards, in-vitro oxidation, and proof-ofconcept analysis of samples (method 3). The experimental conditions for each method are summarized in Table 1.

MS/MS spectra of standards.

A total of 54 and 57 individual oxylipin standards were injected with the methods 1 and 2, respectively, and 28 with both methods. Furthermore, 2 and 6 mixtures of oxylipin standards were injected with the methods 1 and 3, respectively. Altogether, it resulted in 119 files in.raw format. The details of the standards and the composition of the mixtures are available in Supplementary Tables S1, S2.

Analysis of oxPUFA and oxidized oxylipins by LC-MS/MS data dependent acquisition (DDA).

All samples were randomly injected with the method 3 in two batches. Analysis of the first batch consisted of the 40 oxPUFA samples, a pooled quality control sample (QC), which was prepared mixing 5 μL of each oxPUFA sample, and a blank sample both injected each 10th samples. Different DDA inclusion lists were made for each PUFA oxidation calculating the theoretical molecular formulae after adding different levels of oxidation following the known mechanisms of PUFA oxidation. The inclusion lists are available in Supplementary Table S3. The second batch contains the microscaled oxidized 15-HETE, and DMSO-rearranged PGH 1 , and PGH 2 .

Analysis of blood serum and microalgae samples by LC-MS/MS DDA.

The processed blood serum and microalgae samples were injected into the LC-MS/MS system following the method 3. A combine inclusion list of the four different inclusion lists (per PUFA) was used.

NEO-MSMS library building.

The analysis of standards yielded 119 files in.raw of which 56 were from method 1 and 63 from methods 2 and 3. The files were converted to centroided.mzML format using MSconvert38 (version 3.0.23056) for the method 1 files, while ThermoRawFileParser39 version 1.4.2 was used for the Orbitrap IDx files (i.e. methods 2 and 3). This last conversion allowed to remove the fluoranthene calibration masses from the MS1 and MS2 spectra. Subsequently, the MS/MS spectrum for each standard was manually selected using MZmine3 MS spectra visualizer tool (version 3.7.0)40, focusing on the peak apex identified through the extracted ion chromatogram (EIC) with a 3 ppm mass error relative to the theoretical [M-H] precursor. When a MS/MS was extracted from a mixture of standards, the identity of each isobaric feature was confirmed by the relative retention time and specific MS/MS fragments published in our previous works3 or in the mixture datasheet for commercial mixtures. Then, the MS/MS spectra were converted to.mgf format resulting in 195 different spectra of which 131 are unique compounds. This.mgf files were submitted to GNPS libraries8,9,41 employing the 'Batch Upload of Annotated Spectra' workflow (https://ccmsucsd.github.io/GNPSDocumentation/batchupload/). Additionally, another submission was done employing 217 MS/MS.mgf files of commercial standards obtained from Watrous et al. study focused on enzymatic oxylipins7. This building procedure was summarized in Fig. 1.

Classical Molecular network of standards.

Every individual MS/MS spectrum obtained according to the library building section were subjected to classical molecular networking8 using the GNPS platform (https://gnps.ucsd.edu/). The parent mass tolerance and MS/MS fragment ion tolerance were set at 0.02 Da for the analysis. The data were not clustered using MS-Cluster. The resulting network was filtered based on edges, ensuring a cosine score above 0.71 and a minimum of 5 matched peaks. Additionally, edges between two nodes were retained in the network only if each node appeared in the respective top 10 most similar nodes of the other node. To allow for an unlimited number of nodes in a single network, the maximum size of nodes was set to 0 in the connected network. chromatographic threshold = 50%, search minimum in RT range (min) = 0.03, minimum relative height = 1%, minimum absolute height = 2E4, min ratio of peak top/edge = 1, peak duration range (min) = 0.00-1 and minimum scans (data points) = 4. The extracted features of the same PUFA were aligned together using a m/z tolerance of 3 ppm, and retention time tolerance of 0.03 min. The peak list was filtered to retain only features with MS/MS features. All features present in the blank samples have been removed, unless the intensity (peak height) of the feature is 300% more intense than that present in the blank. The alignment was checked with the ISs features which were picked and aligned correctly across samples. The QC data was not used in this alignment approach but provided as a part of this data descriptor for further studies more focused on the quality assurance of semi quantification. All evenmass features have been removed to simplify the studied molecular network. Gap filling is employed to reduce false missing values, using an intensity tolerance of 20%, an m/z tolerance of 3 ppm, and a minimum of 5 scans. Also, an additional feature table was obtained by aligning the feature tables from each PUFA using the same parameters stated before (i.e., all PUFA alignment). The resulting. mgf and.csv files were exported using the built-in "Export/Submit to GNPS/FBMN" module in MZmine3 using the merge MS/MS approach. It was employed with specific parameters to enhance the analysis. Spectra were selected for merging across samples, and the M/Z merge mode utilized a weighted average with the removal of outliers. Intensity merge mode operated by summing intensities. The criteria for merging included an expected mass deviation of 0.001 m/z or 5 ppm, ensuring precision in the spectral combination. A cosine threshold of 70% was set to evaluate the similarity between spectra, and a signal count threshold of 20% was implemented to filter out low-intensity signals. The isolation window offset was set at 0 m/z, and the window width was established at 3 m/z. Finally, the molecular networks were created using the online FBMN workflow42 (version release_28.2) at GNPS platform (https://gnps.ucsd.edu/) with a parent mass tolerance of 0.02 Da and an MS/MS fragment ion tolerance of 0.02 Da. Edges in the network were filtered to have a cosine score above 0.6 and more than 5 matched peaks. Additionally, edges between two nodes were retained only if each node appeared in the respective top 15 most similar nodes of the other and a maximum connected component size of 50.

The spectra in the network were compared against GNPS spectral libraries and our 3 libraries built in this work. Matches between network spectra and library spectra were required to have a cosine score above 0.6 and at least 5 matched peaks. The molecular networking data were analyzed and visualized using Cytoscape (ver. 3.10.0) and was used for the dereplication of new putative NEO-PUFAs studying the fragmentation patterns as described in the Technical Validation section. The construction of the pie charts that will be visualized on the feature based molecular network (FBMN) is based on the peak heights of the corresponding feature in each sample.

Data analysis of oxidized and rearranged oxylipins.

The identification of putative oxylipins from the 15-HETE oxidation and PGH rearrangement experiments was performed by extracting the EICs of specific parent masses on MZmine3 (version 3.7.0) with 3 ppm tolerance and extracting the MS/MS spectra of the integrated features at peak apex. Subsequently, the identification is based on the study of MS/MS data based on known oxylipin fragmentation mechanisms.

Molecular networking of serum, microalgae samples, and oxPUFA.

The LC-MS/MS data of the microalgae and serum samples was processed as well as the oxPUFA samples with MZmine3 using the same parameters. The features were aligned for each type of sample resulting in two pairs of feature tables and the MS/MS files for the serum and microalgae samples, respectively. Then, a molecular network was constructed employing the GNPS classical molecular network workflow using the extracted MS/MS files from the serum, the microalgae, and the 'all oxPUFA' alignment described above. The networking parameters remained consistent with those used for the feature-based molecular network, with the addition of the 'MS cluster OFF' (i.e., it is therefore, a feature-based molecular network) option and library matching against both GNPS and our NEO-MSMS libraries.

Data Records

All the data reported in this article is available in the Zenodo repository under a CC BY 4.0 license as a compressed. zip file with different subfolders43. The folder 'libraries' contains the NEO-MSMS library divided into three.mgf files, i.e., the standards obtained with Q-Exactive Focus (i.e. NEOMSMS_QFocus_lib.mgf), Orbitrap ID-X Tribrid (i.e. NEOMSMS_idX_lib.mgf), and from the Watrous et al. study7 (NEOMSMS_Watrous_lib.mgf). Moreover, the individual.mgf files from each compound was included in three subfolders following the same syntax. The folder 'oxPUFAs' contains the raw.mzML files of the LC-MS/MS analysis and the MZmine3 (version 3.7.0) alignment results (i.e., the feature tables and the extracted.mgf files). The folder 'algae_serum' contains the same type of data from the microalgae and serum samples. The folder 'oxOxylipins' contains the raw.mzML files of the LC-MS/MS analysis of the miniaturized oxidation of HETE, rearrangement of PGH1 and PGH2. Finally, the 'putative_lib´ folder contains the putatively annotated oxylipins in the same structure as 'libraries' folder. All libraries in.mgf format are compatible with GNPS8 and MZmine340. Also, the.mzML files of the different experiments as well as the. mgf files of the standards are available in MASSIVE as a GNPS dataset under CC0 1.0 Universal license44.

Technical Validation

The validation of the molecular networking of NEO-PUFAs was accomplished through four distinct strategies. Firstly, the topology of the molecular network generated using the NEO-MSMS standards as input was examined to determine if the uploaded MS/MS data could highlight structural similarities among the standards present in the dataset.

Next, we generated four feature-based molecular networks for each PUFAs, encompassing all types of oxidation. Additionally, a feature-based network was established, including all four fatty acids, to identify potential compounds based on observed connections within the molecular network from which was created our putative library. Moreover, the examination of oxPUFA data played a crucial role in identifying specific putative compounds which were subsequently added to the putative library.

Finally, the dereplication efficiency of the NEO-MSMS oxylipins dataset implemented in GNPS libraries was estimated by annotating the molecular network obtained from blood serum and microalgae extract, and the oxPUFA data large oxylipins/NEO-PUFAs coverage was revealed by highlighting related oxylipins.

Cartography of classical molecular network of oxylipin standards library.

In Fig. 2 is described a classical molecular network that includes all available standards analyzed on the Orbitrap IDx and the Q-Exactive Focus, presented in circular-shaped nodes, along with the standards extracted from the study by Watrous et al.7, presented in rectangular-shaped nodes. Therefore, this molecular network represents all compounds from the NEO-MSMS library, and their organizational hypotheses which will be used during annotation. Three ways that allow us to classify the compounds can be observed depending on the relationships between structures. Firstly, the compounds are classified by families. For example, it can be observed the F families of isoprostanes45 or prostaglandins colored in cyan and grouped together. The linear compounds (non-cyclic) in orange are linked together by series, while the remaining prostaglandin families of types A, B, J, E, D, K, H, or I are classified separately and presented in violet color.

Secondly, on the other hand, the classification of these compounds is also influenced by the number of hydroxyl groups (OH) present in the molecule, excluding the acidic part. For instance, although 13, 14-dihydro-19-hydroxy-PGE1 belongs to the E family, it is found within the cluster of F families (top right corner). This can be attributed to the presence of three free OH groups in this molecule leading to fragments resembling the F family.

Lastly, the presence of ketone or hydroperoxide forms, indicated by red color contours (middle left corner in violet). Oxylipins/NEO-PUFAs with 18 Da difference will likely be linked in a molecular network if they are either lipid-hydroperoxide and its corresponding ketone. Similarly, because of similarity in fragmentation patterns, it is also the case for D/E-PG derivatives and their corresponding A/J-PG like isomers.

Feature-based molecular networks of oxPUFAs.

NMBHA oxidation.

A FBMN was created for each fatty acid containing the different studied oxidation. As indicated in the key (Fig. 3), we can examine the distribution of the several DHA oxidation performed: the red colour corresponds to oxidation 1 (H2O2), green corresponds to oxidation 2 (H2O2 followed by KOH), yellow and orange represent the first and second fractions of oxidation 3 (V70), while brown represents the only fraction collected with oxidation 4 (V-70 and NMBHA) as the last fraction contaminated with NMBHA was discarded. The squared bold shape are compounds annotated by our NEO-MSMS library.

The FBMN analysis of oxidized DHA, or individually oxidized PUFA, can revealed some rational clustering trends accordingly to families of oxylipins, because whatever the series within a family, a fragmentation pattern remains. For example, the zoomed cluster in Fig. 3, thanks to the library matching using the NEO-MSMS, allowed the annotation of four series of the F4-NeuroP family: 20-F4-NeuroP, 4-F4-NeuroP, 10-F4-NeuroP, and 14-F4-NeuroP. The several detected stereoisomers per series of NeuroPs are represented by the multiple nodes allowing a rapid dereplication knowing the similarity in fragmentations for diastereomeric compounds, but difference in retention time thanks to FBMN. The interconnection of the nodes can be explained by the fragmentation patterns of the F-series isoprostanoids (or PGs) which is well studied in ESI mode (see Supplementary Information Figure S2)46.

Another interesting feature, revealed by the pie charts of such compounds is the NMBHA radical H-atom donor effectiveness at competing with cyclisation processes. The relative quantitative differences in the formation of series of NeuroPs are known to be a direct consequence of further cyclization leading to dioxolane-NeuroPs47. In autoxidative condition the 4-and 20-NeuroPs are the major compounds because they cannot further formed 1, 2dioxolane to the contrary of the other series. However, when NMBHA is used, it reduces the possibility of dioxolane formation, hence the observation of quantitatively higher level of 10 and 14-F4-NeuroPs at the detriment of 4-and 20-F4-NeuroPs. Dereplication and finding "novel" features (Fig. 3) is also indicative of the power of molecular networking approach for finding related metabolites. The few non-annotated nodes in this zoomed cluster, like explained above, are potential putative F-type NeuroP. For example, if we take this unknown node with the same precursor mass as F4t-NeuroP (highlighted with a star), it is relatively easy to find the missing link as the two main fragments (outside the redundant fragments described in Supplementary Information Figure S2) are m/z 113.0608 and m/z 141.0557 indicative of the 7-series47 (Supplementary Information Figure S3). Thus, several compounds have been putatively annotated with a Fprostane ring including DHA, EPA, AA, and ALA precursors.

Phosphite reduction.

Trimethyl phosphite or triphenyl phosphite is known to reduce hydroperoxide derivatives and also interestingly to selectively reduce the isobaric NEO-PUFAs bicyclic-endoperoxides (like PGG) versus the more stable linear endoperoxides (1, 2-dioxolanes)48. It allowed to potentially locate those functions and/or structures (PGG like) in clusters of metabolites (Supplementary Information Figure S4).

All oxPUFA.

As described above, the molecular networking analysis of individual oxidized PUFA is capable of isolating cluster of family of NEO-PUFAs, with the potential of further annotating remaining series. We have established a feature-based molecular network incorporating all the analyzed oxidized PUFAs through the alignment of the four previously used feature tables (mimicking real samples), allowing us to establish new connections between series of different PUFA precursors from the same families. For instance, (Fig. 4, right corner), automatic dereplication using the NEO-MSMS library highlighted the cluster comprising the E-or D-type NEO-EPA (thanks to the identification of PGE3, or its likely isoprostanoid diastereoisomer at m/z 349.2021, orange nodes), 9-E1-PhytoP derivatives (from ALA of m/z 325.2021, violet nodes), as well as E-or D-type DHA derivatives (m/z 375.2177, blue nodes) and A-or J-type EPA metabolites (m/z 331.1915, orange nodes). It is then possible to annotate some of the detected E4-and A3-IsoPs based on their MS/MS fragmentations, as 4-E4-NeuroP and 17-E4-NeuroP (Supplementary Information Figure S5 andS6). Furthermore, 15-A3-IsoP was easily identified as directly connected to PGE3 (almost identical fragmentation pattern, Supplementary Information Figure S7)49.

These inter-family relationships have superseded the intra-family series connections obtained from a single oxidized PUFA. Other clusters have maintained the connection between series of the same family, as is the case in the F3-IsoP, F2-IsoP and F4-NeuroP clusters (Fig. 4).

Structural determination of oxylipin oxidation products.

15-HETE Oxidation.

To retrieve data of previously identified NEO-PUFAs (NMR and/or MS/MS) generated by oxidation of peculiar oxylipins, we miniaturized the original V-70 radical initiated oxidation protocol developed by Rector et al.32. Applied on 15 HETE, it permitted to detect and acquire corresponding diol metabolites, of which 4 were elucidated as: the well-known 5, 15-DiHETE and 8, 15-DiHETE (as their commercially available enzymatic isomers), and the rediscovered 9, 15-DiHETE and 14, 15-DiHETE (Supplementary Information Figures S8-S11). Those NEO-PUFAs represented 4 out of the 7 leukotriene-like metabolites reported (the non-use of NMBHA and the non-reduction procedure could explain the lower number of diols recovered).

PGHs transformation into levuglandins.

The elusive MS/MS data of levuglandins and other iso-levuglandins prompted us to perform the protocol described by Salomon et al. that could smoothly transform PGHs into levuglandins (LGE) via the Kornblum-DeLaMare rearrangement29. Accordingly, as described in that seminal paper, several compounds were identified like the corresponding PGF, PGD, PGE, keto-PG (validated by rt and MS/MS from standards, Supplementary Information Figures S12-S16), and ultimately what we putatively assigned as levuglandin-LGE. The given away fragments for LGE2 are the ones in between the two carbonyl functions (m/z 183.1027, m/z 149.0972, m/z 225.1132, m/z 195.1027 and 165.0921 on Fig. 5). Once more, microscalling PUFAs oxidation or transformation is feasible and allows for the retrieval of MS/MS data of previously discovered NEO-PUFAs missing such valuable information.

Oxylipins/NEO-PUFAs dereplication in blood serum and microalgae samples with oxPUFA and NEO-MSMS.

To assess the NEO-MSMS library's annotation capability with real samples, we initially utilized the 'Spectral Library Search' workflow within GNPS9. We used the.mgf files obtained following the preprocessing of serum and algae samples using MZmine3 described in the Methods section. The matching parameters were 0.6 minimum cosine score, at least 5 matching peaks, 0.02 Da precursor error, and 0.02 Da product ion error. Given these parameters, the results will be as follows:

During the serum analysis, a total of 190 compounds were successfully annotated using the NEO-MS/MS library, as indicated in Supplementary Table S4. Similarly, in the microalgae extract analysis, 138 compounds were annotated, as documented in Supplementary Table S5. These annotations account for 45.2% and 50.5% of the overall annotated compounds for serum and microalgae, respectively.

Following the validation of our library, we established a classical molecular network that incorporated MS/MS data from the aligned oxPUFAs features (see all oxPUFA), serum samples, and algae samples, as illustrated in (Fig. 6). For the sake of reusability of oxPUFA data for the community, we chose classical molecular network workflow since FBMN would come short of utility in this case as the alignment procedure is machine/sample dependent. Our oxPUFA data, depicted by the blue nodes, exhibit strong connections to nodes exclusively found in the serum extracts (depicted in red) and algae (depicted in green), indicating that oxPUFA data is particularly effective in discerning other NEO-PUFAs as the ones already present in oxPUFA data (potentially originating from other fatty acids).

An example from the zoomed-in cluster (Fig. 6) showed different nodes annotated as 11-HETE employing NEO-MSMS library (i.e., the black squares of m/z 319.228 in the Fig. 6) which potentially correspond to different isomers due to the difference on the retention times (max. difference of 0.09 min). These nodes are linked with another two with virtually identical mass spectra (cosine score 0.9929), m/z 347. 259 The position of the hydroxyl group relative to the end of the chain plays a crucial role in connecting compounds of the same family. For example, an OH group at position 11 of a C 20 can easily be linked to a monohydroxyl compound with an OH group at position 13 of a C 22 , or an OH group at position 9 of a C 18 . The study of the MS/MS spectrum clearly demonstrated that the compound with m/z 347.259 corresponds to 13-hydroxy-7Z, 10Z, 14E, 16Z-docosatrienoic acid (13-HDT) a metabolite of adrenic acid (the 16th isomer position could potentially be E). This is supported by the specific fragments at m/z 195 and m/z 223, as shown in Supplementary Information Figure S17.

Regarding the compound with m/z 293.2122 (C 18 H 29 O 3 ), the 9-monohydroxyl of linolenic acid (C 18 H 28 O 2 ) was initially investigated but two additional Da on the specific major fragments led us to consider other isomers of ALA, such as pinolenic acid. The putative fragments of 9-hydroxy-(5Z, 10Z, 12Z)-octadeca-5, 9, 12-trienoic acid would match with the observed spectrum (Supplementary Information Figure S18). The third node with m/z 345.244 must correspond to the 13-monohydroxyl of Osbond acid (13-hydroxy (4Z, 7Z, 10Z, 14E, 16Z)-docosapentaenoic acid), rather than clupanodonic acid, in order for the major fragments to be explained properly (Supplementary Information Figure S19). The two compounds, 13-HDT and the 9-monohydroxyl of pinolenic acid, are in turn connected to one node with masses m/z 321.243 (C20H33O3), likely a monohydroxyl of dihomo-gammalinolenic acid C20H34O2 (Supplementary Information Figure S20). The plausible characteristic fragments of the 11-hydroxy-(8Z, 12E, 14Z)-icosa-8, 11, 14-trienoic acid correspond perfectly to those observed in the spectrum.

These 'novel oxylipins/NEO-PUFAs' arising from enzymatic or non-enzymatic processes would be completely overlooked if only a targeted analysis is to be performed, questioning the utility of adapting a targeted MRM solution based on oxPUFA data50. The use of oxPUFA data along with our NEO-MSMS library can be highly valuable, both for annotating compounds found in biological matrices and for connecting new structures to discover potential novel biomarkers. The very precise nature of OxPUFA data also likely suggest its use for machine-learning based annotation51.

Dereplication of the NEO-MSMS against the "putative Watrous oxylipins" available on the MASSIVE repository.

In the Watrous studies7,52, more than 500 "unknown or putative oxylipins" were discovered, and as initially hypothesized in this manuscript and one of the purpose for this NEO-MSMS library, we believed that some of them could be related to NEO-PUFAs. We subsequently reanalyzed the data presented by Watrous et al. in 2019 through a conventional molecular network, using the same parameters and incorporating our library NEO-MSMS. However, the MS/MS files available present a mass error that can exceed 0.04 m/z and could not be used to match lipid datasets for predicting known fragmentations (simple loss of water or CO 2 are already off target to match any formulae of oxylipins). Nevertheless, the power of molecular networking is that by increasing the mass error tolerance to 0.1 instead of 0.02, we could carry out dereplication. However, still due to the complexity of further analysis of the data, we specially focused on three specific oxylipins; i.e, oxylipin 1, 2, and 3 (named as such in the original text) which showed significant changes after one-year PUFA n-3 treatment (VITAL study) and associated with clinical lipid and inflammatory biomarkers52. We also looked at five other unidentified putative compounds from the original Watrous et al. study which were frequently found to be associated with multiple correlates of inflammation, while other putative compounds, were specifically linked to age, BMI, or CRP levels, suggesting both universal and specific inflammation mediators7.

As described (in the Supplementary Information Figs. S21), 6 out of the 8 putatively assigned oxylipins (vide supra) could be annotated with great confidence as NEO-PUFA (isomer of 5-HETE, 9-epi-9-F1t-PhytoP, isomer of 20-HDHA, 11-hydroxy-7E, 9Z, 13Z, 16Zdocosatrienoic acid (11-HDT), 13-hydroxy-7E, 10E, 14E, 16Z-docosatrienoic acid (13-HDT) and 14, 17-DiHEPE) and not as enzymatic oxylipins, thanks to the NEO-MSMS associated with the library building of GNPS and the molecular networking facility of GNPS using oxPUFA data.

We validate that NEO-MSMS and oxPUFA data could help reinterpreting the previously identified families of NEO-PUFAs of which data were not collected at the time. It could also help the isolation of formerly unreported metabolites.

Usage Notes

The NEO-MSMS library can be used directly in the GNPS environment for the different molecular networking analysis. Also, it is available as a set of.mgf files compatible with MZmine3 libraries. oxPUFA files could be used for conducting classical molecular networking analysis with another samples as we demonstrated for serum and algae samples and dereplicate oxylipins. Additionally, the oxPUFA files will also be located in the MASSIVE repository as GNPS dataset allowing their use with GNPS MASST53. With this tool, the oxPUFA data can be re-analyzed and used for the annotation of unknowns as putative NEO-PUFA in future lipidomic studies with special value for the novel separation approaches employing sophisticated chiral LC54 or ion mobility55,56.

Feature-based molecular

  networking of oxPUFAs. The LC-MS/MS data files associated with oxPUFAs were converted from the.raw data format to centroided.mzML files using the ThermoRawFileParser version 1.4.239. Subsequently, the.mzML file was processed further using MZmine3 (version 3.7.0)40. Mass detections were conducted with a noise level threshold of 2E4 in MS1 and 2E3 in MS/MS. The ADAP chromatogram builder utilized a minimum group size of 4 scans, a group intensity threshold of 5E3, a minimum highest intensity of 1E4, and an m/z tolerance of 3 ppm. Chromatogram deconvolution employed the Local Minimum Search algorithm with the following settings:

  (C 22 H 35 O 3 ), which could correspond precisely to a monohydroxyl derivative of a fatty acid with the formula C 22 H 36 O 2 . Similarly, the node with m/z 345.244 (C 22 H 34 O 3 ) may correspond to a monohydroxyl derivative of C 22 H 34 O 2 , and m/z 293.2122 (C 18 H 29 O 3 ) to the monohydroxyl derivative of C 18 H 28 O 2 .

Code availability

No custom code has been used in the generation of this work.


Shubhneet Thind

Dhésmon Lima

Evan Booy

Dao Trinh

Sean Mckenna

Dr Sabine Kuss
email: sabine.kuss@umanitoba.ca

Cytochrome c oxidase deficiency detection in human fibroblasts using scanning electrochemical microscopy

Keywords: cytochrome c oxidase deficiency, scanning electrochemical microscopy, single-cell analysis, electrochemistry

Cytochrome C Oxidase Deficiency (COXD) is an inherited disorder characterized by the absence or mutation in the genes encoding for the cytochrome c oxidase protein (COX). COX deficiency results in severe muscle weakness, heart, liver, and kidney disorders, as well as brain damage in infants and adolescents, leading to death in many cases. With no cure for this disorder, finding an efficient, inexpensive, and early means of diagnosis is essential to minimize symptoms and long-term disabilities. Furthermore, muscle biopsy, the traditional detection method, is invasive, expensive, and time-consuming. This study demonstrates the applicability of scanning electrochemical microscopy to quantify COX activity in living human fibroblast cells. Taking advantage of the interaction between the redox mediator N,N,N',N'-tetramethyl-para-phenylene-diamine, and COX, the enzymatic activity was successfully quantified by monitoring current changes using a platinum microelectrode and determining the apparent heterogeneous rate constant k0 using numerical modeling. This study provides a foundation for developing a new diagnostic method for detecting COXD in infants, which has the potential to increase treatment effectiveness and improve the quality of life of affected individuals.

Significance Statement

Cytochrome C Oxidase deficiency (COXD) is an incurable mitochondrial disease, but symptoms in young infants can be managed if an early diagnosis is made, improving the quality of life of affected individuals. Currently, this disorder is diagnosed through muscle biopsies which are invasive, expensive, and time-consuming, leading to a delay in diagnosis. Our study proposes the quantification of cytochrome c oxidase (COX) activity in living fibroblasts using Scanning Electrochemical Microscopy (SECM) as an indicator for the quick, non-invasive, and accurate diagnosis of COXD. Our results will form the basis for the future development of a biosensor for the routine detection of COXD in tissue samples, therefore benefiting healthcare systems and newborns around the world.

Main Text

Introduction

Cytochrome c oxidase (COX), also known as Complex IV, is a multimeric protein comprised of fourteen subunits that act as the terminal electron acceptor of the electron transport chain, which is responsible for coupling oxidative phosphorylation with ATP production [START_REF] Timón-Gómez | Mitochondrial cytochrome c oxidase biogenesis: Recent developments[END_REF][START_REF] Nelson | Oxidative Phosphorylation and Photophosphorylation[END_REF]. Found in the inner membrane of the mammalian mitochondria [START_REF] Nelson | Oxidative Phosphorylation and Photophosphorylation[END_REF], three of the subunits of this transmembrane protein are encoded by mitochondrial DNA while the others are encoded by nuclear DNA [START_REF] Horng | Specific copper transfer from the Cox17 metallochaperone to both Sco1 and Cox11 in the assembly of yeast cytochrome c oxidase[END_REF]. Mutations in mitochondrial or nuclear genes encoding COX lead to the incorrect assembly of COX, resulting in functional defects which cause a rare heterogenous disorder called cytochrome c oxidase deficiency (COXD) [START_REF] Brischigliaro | Cytochrome c oxidase deficiency[END_REF]. This disorder dramatically affects vital organs such as the heart, brain, liver, and skeletal muscles, causing a range of degenerative diseases, from lethal infantile myopathy to Leigh Syndrome and Hepatic Encephalopathy [START_REF] Brischigliaro | Cytochrome c oxidase deficiency[END_REF][START_REF] Wallace | Mitochondrial defects in cardiomyopathy and neuromuscular disease[END_REF][START_REF] Fontanesi | Mitochondrial Cytochrome c Oxidase Assembly in Health and Human Diseases[END_REF]. Being the most prevalent among all mitochondrial disorders (30% of the cases) [START_REF] Valnot | Mutations of the SCO1 gene in mitochondrial cytochrome c oxidase deficiency with neonatal-onset hepatic failure and encephalopathy[END_REF][START_REF] Von Kleist-Retzow | A High Rate ( 20%-30%) of Parental Consanguinity in Cytochrome-Oxidase Deficiency[END_REF], which affect 1 in 5000 people worldwide [START_REF] Gorman | Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease[END_REF], COXD is often fatal in childhood, with only a few individuals surviving adolescence [START_REF] Brischigliaro | Cytochrome c oxidase deficiency[END_REF][START_REF] Shoubridge | Cytochrome c oxidase deficiency[END_REF]. Mutations in thirty possible nuclear and mitochondrial genes are known to cause COXD [START_REF] Brischigliaro | Cytochrome c oxidase deficiency[END_REF], which includes alterations in the SCO1 nuclear-encoded gene. SCO1 encodes the SCO1 metallochaperone protein [START_REF] Stiburek | Loss of function of Sco1 and its interaction with cytochrome c oxidase[END_REF][START_REF] Leary | Human SCO1 and SCO2 have independent, cooperative functions in copper delivery to cytochrome c oxidase[END_REF], which mediates copper transport from subunit COX17 to COX2, allowing the assembly of the CuA copper site in COX and ultimately enabling the proper biosynthesis of the protein [START_REF] Horng | Specific copper transfer from the Cox17 metallochaperone to both Sco1 and Cox11 in the assembly of yeast cytochrome c oxidase[END_REF][START_REF] Stiburek | Loss of function of Sco1 and its interaction with cytochrome c oxidase[END_REF]. Therefore, mutations in SCO1 indirectly impair the proper assembly of COX, leading to the biosynthesis of nonfunctional COX molecules. COXD caused by SCO1 mutation results in clinical phenotypes such as hepatic failure [START_REF] Shoubridge | Cytochrome c oxidase deficiency[END_REF], fatal encephalopathy (4), and ketoacidosis [START_REF] Valnot | Mutations of the SCO1 gene in mitochondrial cytochrome c oxidase deficiency with neonatal-onset hepatic failure and encephalopathy[END_REF].

Currently, COXD is diagnosed by collecting skin and muscle biopsy samples from patients which are subject to a variety of enzyme assays and histochemical methods including spectrophotometric, immunofluorescent, and cytochrome c oxidase/succinate dehydrogenase double-labeling (COX/SDH) assays [START_REF] Chakraborthy | Photo_Human Cytochrome Oxidase Deficiency[END_REF][START_REF] Parikh | Diagnosis and management of mitochondrial disease: a consensus statement from the Mitochondrial Medicine Society[END_REF][START_REF] Frazier | Biochemical Analyses of the Electron Transport Chain Complexes by Spectrophotometry[END_REF][START_REF] Simard | A novel histochemistry assay to assess and quantify focal cytochrome c oxidase deficiency[END_REF][START_REF] Wortmann | A Guideline for the Diagnosis of Pediatric Mitochondrial Disease: The Value of Muscle and Skin Biopsies in the Genetics Era[END_REF]. These methods present several drawbacks. For instance, COX/SDH is often limited by its inability to detect COXD at low levels due to this method's dependence on staining [START_REF] Murphy | Cytochrome c oxidase-intermediate fibres: Importance in understanding the pathogenesis and treatment of mitochondrial myopathy[END_REF]. Immunofluorescencebased methods require expensive antibodies to detect COX activity, which significantly increases the cost of the technique [START_REF] Simard | A novel histochemistry assay to assess and quantify focal cytochrome c oxidase deficiency[END_REF][START_REF] Grünewald | Quantitative quadruple-label immunofluorescence of mitochondrial and cytoplasmic proteins in single neurons from human midbrain tissue[END_REF]. Additionally, these assays are labor-intensive, time-consuming, and often require invasive muscle biopsies from young patients [START_REF] Simard | A novel histochemistry assay to assess and quantify focal cytochrome c oxidase deficiency[END_REF][START_REF] Murphy | Cytochrome c oxidase-intermediate fibres: Importance in understanding the pathogenesis and treatment of mitochondrial myopathy[END_REF][START_REF] Ross | Visualization of mitochondrial respiratory function using cytochrome C oxidase/succinate dehydrogenase (COX/SDH) double-labeling histochemistry[END_REF], as non-invasive means of COX diagnosis are currently non-existent [START_REF] Parikh | Diagnosis and management of mitochondrial disease: a consensus statement from the Mitochondrial Medicine Society[END_REF]. The development of alternative sensitive methods that are capable of diagnosing COXD inexpensively, quickly, and non-invasively, is therefore crucial.

Electrochemical methods have revealed an outstanding potential to be used as diagnostic assays to detect many disease biomarkers. Specifically, scanning electrochemical microscopy (SECM) stands out as an innovative technique that can detect trace levels of biomolecules with high spatial and temporal resolution. During SECM, while recording its lateral tip position, a microelectrode is scanned across an underlying surface monitoring an electrochemical current. Unlike other scanning probe techniques, SECM not only reveals the topography of samples in a solution but also enables the detection and visualization of local variations in the electrochemical reactivity of the investigated sample. The high sensitivity of this method, its non-invasive nature, and the micrometric dimensions of the sensor make SECM a particularly attractive technique to study biological samples, such as living cells and tissues [START_REF] Polcari | Scanning Electrochemical Microscopy: A Comprehensive Review of Experimental Parameters from 1989 to 2015[END_REF][START_REF] Filice | Analysing single live cells by scanning electrochemical microscopy[END_REF][START_REF] Beaulieu | Biological scanning electrochemical microscopy and its application to live cell studies[END_REF]. In fact, several promising biological applications of SECM have been described, including the study and detection of human cancer [START_REF] Kuss | Assessment of multidrug resistance on cell coculture patterns using scanning electrochemical microscopy[END_REF][START_REF] Kuss | High-Speed Scanning Electrochemical Microscopy Method for Substrate Kinetic Determination: Application to Live Cell Imaging in Human Cancer[END_REF][START_REF] Darvishi | Tape-Stripping Electrochemical Detection of Melanoma[END_REF], and bacterial metabolism [START_REF] Connell | Real-time monitoring of quorum sensing in 3D-printed bacterial aggregates using scanning electrochemical microscopy[END_REF][START_REF] Jayathilake | Glucose Microsensor with Covalently Immobilized Glucose Oxidase for Probing Bacterial Glucose Uptake by Scanning Electrochemical Microscopy[END_REF][START_REF] Zhou | Bacterial Biofilm Probed by Scanning Electrochemical Microscopy: A Review[END_REF]. The exceptional features of SECM make it an ideal tool to detect new cellular biomarkers and cell metabolites at the single-cell level [START_REF] Thomas | Single-cell scanning photoelectrochemical microscopy using micro-optical-ring electrodes[END_REF][START_REF] Zhao | Profiling H2O2 from single COS-7 cells by means of scanning electrochemical microscopy[END_REF] that can later be transferred onto portable, cost-efficient, and userfriendly biosensing platforms.

In this study, we propose a sensitive electroanalytical method based on SECM to quantify COX activity in living primary fibroblasts, which are known for being a model cell line used to study mitochondrial disorders associated with COX [START_REF] Salvador-Severo | Mitochondrial proteomic profile of complex IV deficiency fibroblasts: rearrangement of oxidative phosphorylation complex/supercomplex and other metabolic pathways[END_REF]. Our detection approach uses the redox mediator N,N,N',N'-tetramethyl-para-phenylene-diamine (TMPD). TMPD is commonly used as the substrate in the oxidase test to determine the presence of cytochrome c oxidases in bacteria [START_REF] Bascomb | Enzyme Tests in Bacterial Identification[END_REF][START_REF] Jurtshuk | Tetramethyl-p-phenylenediamine oxidase reaction in Azotobacter vinelandii[END_REF], and has been previously employed to quantify bacterial cytochrome c oxidase activity using electrochemistry [START_REF] Kuss | Electrochemical recognition and quantification of cytochrome c expression in Bacillus subtilis and aerobe/anaerobe Escherichia coli using N, N, N ′, N ′-tetramethyl-paraphenylene-diamine (TMPD)[END_REF].

Herein, SECM measures fibroblasts' ability to oxidize TMPD to TMPD +• , which is mediated by COX. The redox species diffuse through the mitochondrial outer membrane and cross the lipid cell bilayer to reach the microelectrode, where it is reduced. The generated current response is a convoluted signal that is influenced by the cell's reactivity, but also by the cell's topography [START_REF] Bard | Scanning Electrochemical Microscopy. Introduction and Principles[END_REF]. To decouple the effect of topography and reactivity on the detected current signal, a numerical modeling approach described previously is employed [START_REF] Kuss | High-Speed Scanning Electrochemical Microscopy Method for Substrate Kinetic Determination: Application to Live Cell Imaging in Human Cancer[END_REF][START_REF] Kuss | High-Speed Scanning Electrochemical Microscopy Method for Substrate Kinetic Determination: Method and Theory[END_REF]. This methodology enables the extraction of the apparent heterogeneous rate constant and, thereby quantifying COX activity in living fibroblasts. This is the first report on an electrochemical method devoted to investigating COXD, and the presented results will serve as a foundation for the future development of a portable biosensor for COXD diagnosis.

Results and Discussion

COX redox mechanism and TMPD electrochemistry

As seen in Scheme 1A, under normal aerobic conditions, COX catalyzes the electron transfer from reduced cytochrome c to molecular oxygen, resulting in water and oxidized cytochrome c, while transporting one proton per electron from the matrix to the intermembranous space [START_REF] Nelson | Oxidative Phosphorylation and Photophosphorylation[END_REF]. Simultaneously, cytochrome c oxidoreductase or complex III (CRE) pumps two protons per electron while catalyzing electron transfer from ubiquinol (QH2) to oxidized cytochrome c, restoring cytochrome c to its reduced form. Cytochrome c can only transfer one electron at a time; hence, to produce one water molecule, cytochrome c must undergo reduction twice (2).

To enable the electrochemical detection of COX activity using SECM, we used TMPD as the redox mediator due to its well-known interaction with the protein [START_REF] Bascomb | Enzyme Tests in Bacterial Identification[END_REF][START_REF] Jurtshuk | Tetramethyl-p-phenylenediamine oxidase reaction in Azotobacter vinelandii[END_REF][START_REF] Kuss | Electrochemical recognition and quantification of cytochrome c expression in Bacillus subtilis and aerobe/anaerobe Escherichia coli using N, N, N ′, N ′-tetramethyl-paraphenylene-diamine (TMPD)[END_REF]. In the presence of TMPD concentrations higher than 0.4 mM, electron transfer reactions can occur directly from TMPD to COX, without the involvement of cytochrome c [START_REF] Villani | In vivo control of respiration by cytochrome c oxidase in wild-type and mitochondrial DNA mutation-carrying human cells[END_REF][START_REF] Nicholls | Respiratory Chains[END_REF]. Scheme 1B proposes the cellular uptake of the membrane-permeable redox mediator TMPD [START_REF] Villani | In vivo control of respiration by cytochrome c oxidase in wild-type and mitochondrial DNA mutation-carrying human cells[END_REF]. TMPD crosses the outer cell membrane barrier, as well as the mitochondrial outer membrane, interacting with COX located in the inner mitochondrial membrane.

TMPD undergoes oxidization to TMPD +• , transferring one electron to COX to reduce oxygen to produce water [START_REF] Salabei | Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis[END_REF]. Just as in the case of cytochrome c, two TMPD molecules are required to produce one water molecule. COX can transport one proton per electron into the intermembranous space, allowing ATP synthesis to continue through the ATP synthase. Once oxidized, TMPD +• passes through the outer mitochondrial membrane and the cell plasma membrane to interact with the surface of the microelectrode as it passes over the cell, as seen in Scheme 1C. A reduction potential, applied at the electrode reduces TMPD +• back to TMPD, creating a feedback loop, which produces an increase in electrochemical current during SECM imaging.

To understand TMPD's role as a redox mediator for single-cell SECM studies, it was electrochemically characterized using cyclic voltammetry. TMPD undergoes two electron transfer steps, generating TMPD +• first, and TMPD 2+ in the second electron transfer step [START_REF] Brownson | The Handbook of Graphene Electrochemistry[END_REF]. As shown in Figure 1A, TMPD oxidizes at +200 mV to produce TMPD +• and is subsequently oxidized to TMPD 2+ at +600 mV. Reduction potentials are observed at +200 mV and -200 mV. The effect of electrolyte composition was studied in DMEM cell media, in the presence and the absence of FBS, and 0.1 M KCl (Figure S1). Although similar voltammetric profiles were observed for TMPD in DMEM media with and without FBS (Figure S1), all SECM experiments were performed in the absence of serum, because of potential electrode fouling over time [START_REF] Kuss | Assessing multidrug resistance protein 1-mediated function in cancer cell multidrug resistance by scanning electrochemical microscopy and flow cytometry[END_REF]. To avoid the formation of the unstable TMPD 2+ , SECM measurements on living cells were performed by applying a potential at the microelectrode that corresponds to the first electron transfer step (TMPD to TMPD +• and vice versa).

Electrochemical monitoring of TMPD interaction with living fibroblasts

The two fibroblast cell lines used in this work included a control cell line (Control 65) of healthy fibroblasts, capable of expressing a fully assembled and functional COX enzyme. SCO1 cells carry the SCO1 gene mutation that impairs the proper biosynthesis of COX [START_REF] Horng | Specific copper transfer from the Cox17 metallochaperone to both Sco1 and Cox11 in the assembly of yeast cytochrome c oxidase[END_REF][START_REF] Stiburek | Loss of function of Sco1 and its interaction with cytochrome c oxidase[END_REF]. Both cell types present a similar morphology, characterized by an elongated shape with a fibrous aspect (Figures 1B andC). To validate the cell lines of choice, expression levels of fully assembled COX and SCO1 proteins in the Control and SCO1 cell lines were quantified by Western blot analysis. The SDS-PAGE result displayed in Figure 2A confirms the reduced level of SCO1 protein (29 kDa) in SCO1 cells. VDAC1 (35 kDa) was used as a loading control and was detected equally in both cell lines.

Accordingly, the BN-PAGE results (Figure 2B) show that cells carrying the SCO1 mutation present reduced levels of fully assembled COX due to decreased levels of SCO1 protein Prior to SECM measurements, cells were exposed to DMEM basic media (no FBS) in the presence of 1 mM TMPD for 15 min. Cell viability was not affected by TMPD exposure according to a Trypan Blue exclusion cell viability assay in the literature, where it has been reported previously that cell exposure to TMPD for 15 min does not affect viability [START_REF] Bell | The Qo site of the mitochondrial complex III is required for the transduction of hypoxic signaling via reactive oxygen species production[END_REF]. However, the absence of FBS in the cell culture media is known to affect both cell viability and morphology [START_REF] Kuss | Assessing multidrug resistance protein 1-mediated function in cancer cell multidrug resistance by scanning electrochemical microscopy and flow cytometry[END_REF]. By conducting a cell viability study over 90 min, we were able to confirm that the viability and morphology of Control and SCO1 fibroblast lines remained unaffected for the first 60 min (Figure S2), and no external signs of stress were observed through the SECM-integrated optical microscope. This ensures that fibroblasts remained viable during all SECM line scans.

Three-dimensional SECM images (Figure 3) of single Control and SCO1 fibroblasts show a differential current behavior between the two cell lines, qualitatively. In this representative example of an experimental data set, larger current signals from a Control cell (Figures 3C andD) can be seen compared to the current obtained from a SCO1 cell (Figures 3A andB). Three-dimensional images were obtained in TMPD at 50 µm s -1 scan velocity, using a microelectrode biased at the potentials of +200 mV (oxidations of TMPD) or -200 mV (reduction of TMPD). It should be noted that a solution of TMPD prepared in the presence of oxygen naturally presents a ratio of 3:1 of reduced and oxidized TMPD [START_REF] Kuss | Electrochemical recognition and quantification of cytochrome c expression in Bacillus subtilis and aerobe/anaerobe Escherichia coli using N, N, N ′, N ′-tetramethyl-paraphenylene-diamine (TMPD)[END_REF], respectively. When a reduction potential of -200 mV is applied, the electrode detects the ability of the living cells to oxidize TMPD to TMPD+•, which is reduced back to TMPD at the microelectrode (Scheme 1). During SECM imaging of living cells, the measured signal is always a convolution of topographical contributions and reactivity-related signals [START_REF] Kuss | High-Speed Scanning Electrochemical Microscopy Method for Substrate Kinetic Determination: Application to Live Cell Imaging in Human Cancer[END_REF][START_REF] Kuss | High-Speed Scanning Electrochemical Microscopy Method for Substrate Kinetic Determination: Method and Theory[END_REF]. Topographical contributions result in a decrease in current, because of the hindered diffusion of the redox active species towards the electrode due to the topography of the cell. Reactivity describes the regeneration of the redox mediator by the living cell. As shown in Figures 3A andC, as the microelectrode passes over the cells, a clear increase in the electrochemical current signal was observed.

In this case, the reactivity of the cells is able to overcompensate for the topographical contributions, resulting in an overall increase in current when the electrode passes the cell located on the petri dish. Similarly, when an oxidation potential of +200 mV is employed (Figures 3B andD), the electrode detects the ability of the living cells to reduce TMPD +• to TMPD, which is then oxidized to TMPD +• at the microelectrode. In this case, the reactivity of the cells is not able to overcompensate for the contributions from the cell topography, resulting in an overall decrease in current when the electrode scans across the living cells (Figure 3B andD). Interestingly, depending on the applied potential at the microelectrode, SECM is able to monitor the activity of both cytochrome oxidase and reductase.

However, the qualitative assessment of currents is inaccurate and can be misleading as cells of different shapes and sizes will have differential contributions to the measured electrochemical current from topography and reactivity. Numerical modeling is an effective and accurate tool to decouple topography from reactivity and extract an apparent heterogeneous rate constant k0, which indicates the cell's ability to regenerate a redox mediator in solution [START_REF] Kuss | High-Speed Scanning Electrochemical Microscopy Method for Substrate Kinetic Determination: Application to Live Cell Imaging in Human Cancer[END_REF][START_REF] Kuss | High-Speed Scanning Electrochemical Microscopy Method for Substrate Kinetic Determination: Method and Theory[END_REF][START_REF] Kuss | Forced convection during scanning electrochemical microscopy imaging over living cells: Effect of topographies and kinetics on the microelectrode current[END_REF]. Figure 4 demonstrates SECM line scans in TMPD across Control and SCO1 cells at varying scan rates (10 to 100 µm s -1 ). It is evident that, as the scan velocity is increased, the current measured at the microelectrode also increases because of greater mass transport caused by forced convection [START_REF] Kuss | High-Speed Scanning Electrochemical Microscopy Method for Substrate Kinetic Determination: Method and Theory[END_REF]. Thereby, electrochemical currents that originate from contributions by topography are more susceptible to scan velocity changes, than contributions from reactivity. This forms the basis of the previously reported numerical model, capable of decoupling topography from reactivity [START_REF] Kuss | High-Speed Scanning Electrochemical Microscopy Method for Substrate Kinetic Determination: Application to Live Cell Imaging in Human Cancer[END_REF].

Extraction of the apparent heterogeneous rate constant (k0)

Using numerical modeling, TMPD oxidation and TMPD +• reduction occurring at the microelectrode after interaction with COX and cytochrome c reductase (CRE) in living fibroblasts can be utilized to extract the apparent heterogeneous rate constant (k0), representing the biological cell kinetics. This mathematical approach was described in detail in previous work [START_REF] Kuss | High-Speed Scanning Electrochemical Microscopy Method for Substrate Kinetic Determination: Method and Theory[END_REF][START_REF] Kuss | Forced convection during scanning electrochemical microscopy imaging over living cells: Effect of topographies and kinetics on the microelectrode current[END_REF], and is based on the extraction of a substrate's kinetic rate from electrochemical data obtained using SECM constant height imaging at slow scan velocities (<100 µm s -1 ). The numerical COMSOL model considers experimental parameters such as the microelectrode size, steady state current, scan velocity, diffusion parameters of redox species, as well as cellular features, including the cell's shape and size to quantify the redox environment of a cell. This mathematical approach fits the theoretical results from the substrate kinetic rates varying from 10 -8 m s -1 to 10 -1 m s -1 to the normalized current detected at the microelectrode as the function of scan velocities.

Because the microelectrode current depends on the scan velocity of the tip, the diffusion coefficient, and the tip-to-sample distance, the kinetic rates can be extracted from the normalized shear Peclet number (Ps) [START_REF] Kuss | High-Speed Scanning Electrochemical Microscopy Method for Substrate Kinetic Determination: Method and Theory[END_REF].

A visualization of the electrode and fluid movement within the simulation is presented in Figure S3. When a microelectrode passes a living cell, the cell-surrounding fluid is forced to pass in between the electrode tip and the body of the cell. Because of the limited amount of space for the fluid to pass, the concentration of the redox species in between the cell is temporarily increased. The height of a cell thereby affects the measured current more than the reactivity of the cell, which remains constant. The faster the scan velocity, the more fluid is pushed between the microelectrode and the cell. By simulating the scanning profiles for several scan velocities, an average kinetic rate k0 can be extracted. Figure 5 shows the normalized tip current as a function of Ps for the Control and SCO1 fibroblasts (Figures 5A and5C, respectively). For both COX and CRE activity, the SCO1 values are lower than the Control values, confirming slower kinetic rates in the SCO1 fibroblasts. These kinetic rates can be determined by fitting the normalized current on the simulation curve for both COX activity (Figure 5B) and CRE activity (Figure 5D). Two-sample two-tailed t-tests performed with the results showed that the difference in the average k0 values for COX activity between Control and SCO1 fibroblasts is statistically significant (P < 0.10), whereas it was found to be non-significant when the CRE activity was analyzed (P > 0.10). These results are in clear agreement with the phenotype of the cells analyzed in this study. The mutation in the SCO1 gene in SCO1 fibroblasts affects the biosynthesis of COX, with no direct effect on the expression of the CRE enzyme. However, the observable trend of reduced activity of CRE in SCO1 cells is not surprising as the activities of the enzyme complexes in the electron transport chain are intrinsically connected [START_REF] Vercellino | The assembly, regulation and function of the mitochondrial respiratory chain[END_REF][START_REF] Brzezinski | Structure and Mechanism of Respiratory III-IV Supercomplexes in Bioenergetic Membranes[END_REF]. The statistically significant difference in k0 between Control and SCO1 cells with regards to COX activity is, therefore, a robust indicator that cellular phenotypes of COXD can be accurately and quantitatively monitored using SECM. In this study, we present SECM as a promising and effective method to detect and quantify COX activity in living fibroblasts. The presented electroanalytical approach was based on quantifying current variations resulting from the interaction of the redox mediator TMPD with living cells, followed by extracting an apparent heterogeneous rate constant for the turnover of TMPD/TMPD +• by single cells using numerical modeling. The study of the cell reaction kinetics allowed us to successfully decouple the effects of cell reactivity and cell topography on the overall bioelectrochemical response, hence giving insight into the differential COX activity between Control and SCO1 deficient cells. TMPD/TMPD +• regeneration was found to be considerably slower in the SCO1 cell line compared to healthy control cells, making this SECM approach a reliable method for COX activity detection in human cells. The insight generated in this proof-of-concept study can be further employed in the development of an electrochemical biosensor for COXD clinical diagnostics. Moreover, our study confirms the ability of SECM to identify new biomarkers for diseases and pave the path for the development of new biomedical detection assays.

Conclusions

Material and Methods

All chemicals were purchased from Sigma-Aldrich (Canada), and all media and materials for cell culture and related assays were purchased from Thermo-Fisher Scientific (USA) unless otherwise stated.

Cell Culture and sample preparation for SECM analysis

Control and SCO1 skin fibroblast cell lines were provided by Dr. Eric Shoubridge (McGill University, Canada). These two primary immortalized cell lines were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), in tissue culture flasks at 37 ºC and 5% CO2. After reaching 80-90% confluency, cells were harvested. To this end, cells were washed with 0.0067 M phosphate-buffered saline (PBS, pH 7.0 at 37 ºC) and detached from the surface of the flask using 0.05% trypsin-EDTA solution at 37 ºC. For SECM analysis, around 40,000 cells were seeded in 10 mm x 35 mm Petri dishes and incubated overnight under the conditions described above.

Western blot assay

Following procedures previously described [START_REF] Leary | Human SCO1 and SCO2 have independent, cooperative functions in copper delivery to cytochrome c oxidase[END_REF][START_REF] Klement | Analysis of Oxidative Phosphorylation Complexes in Cultured Human Fibroblasts and Amniocytes by Blue-Native-Electrophoresis Using Mitoplasts Isolated with the Help of Digitonin[END_REF][START_REF] Schägger | Native electrophoresis for isolation of mitochondrial oxidative phosphorylation protein complexes[END_REF], fully assembled COX was detected by subjecting mitoplasts from Control and SCO1 fibroblasts to blue native page gel electrophoresis (BN-PAGE). Mitoplasts were initially isolated using a mitochondrial isolation kit (89874, Thermo-Fisher Scientific). For BN-PAGE analysis, sedimented mitochondria were resuspended in 0.75 M aminocaproic acid and 50 mM Bis-Tris buffer at pH 7. 10% n-dodecyl-B-D-maltopyranoside was then added to the suspension and incubated on ice for 30 minutes. The mixture was centrifuged at 16,000 x g for 30 minutes before 5% Coomassie Brilliant Blue stain in 0.5 M aminocaproic acid was added to the supernatant. For first dimension separation, a 6-15% native polyacrylamide gradient gel was used. The blot was subjected to antibodies against COX (ab14744, Abcam, Cambridge, UK) and loading control complex I (ab110412, Abcam, Cambridge, UK). SDS-PAGE was performed as previously described by Booy et al. [START_REF] Booy | The RNA helicase RHAU (DHX36) unwinds a G4-quadruplex in human telomerase RNA and promotes the formation of the P1 helix template boundary[END_REF], to detect SCO1 protein in Control and SCO1 fibroblasts. This blot was subjected to antibodies against SCO1 (PA554460 Invitrogen, MA, USA) and loading control VDAC1 (MABN504 MilliporeSigma, MA, USA). HRP-conjugated goat anti-rabbit (12348) and HRP-conjugated goat antimouse (12349) antibodies (Millipore Sigma) were used for chemiluminescent detection.

Proteins were visualized using a ChemiDoc System (Bio Rad, CA, USA).

Cell viability assay

To determine the viability of fibroblasts in the presence of TMPD, Trypan Blue Exclusion assays were conducted for Control and SCO1 cell lines. Cells were exposed to 1 mM TMPD in basic DMEM media (no FBS) for varying time intervals of 10, 30, 60, and 90 minutes. The medium was removed, cells were washed with PBS, and then harvested after treatment with trypsin. Subsequently, fibroblasts were incubated in the presence of Trypan Blue (1:1) at room temperature for 3 minutes [START_REF] Strober | Trypan Blue Exclusion Test of Cell Viability[END_REF]. Percent cell viability was determined using a Countess 2 Automated Cell Counter (Thermo Scientific, USA).

Scheme 1 .

 1 Scheme 1. Schematic representation of cellular and electrochemical reactions. (A) COX (violet) and cytochrome c reductase (orange) complexes under normal aerobic conditions in a mammalian cell. (B) SECM measurements at 12 μm above the surface of a single fibroblast cell in 1 mM TMPD in feedback mode (C). Proposed mechanism of Complex IV under TMPD concentration > 0.4 mM, TMPD (T), TMPD + (T +• ), OMM: Outer mitochondrial membrane, IMS: Inter-membrane space, IMM: Inner mitochondrial membrane, MM: Mitochondrial matrix.

Fig. 1 .

 1 Fig. 1. TMPD electrochemistry and cell morphology. (A) Voltametric profile of N,N,N′,N′-tetramethyl-paraphenylenediamine (TMPD) on a bare 25-μm platinum microelectrode. Reduction and oxidation potentials of TMPD are shown by red dotted lines (respectively). (B) Optical micrographs of Control and (C) SCO1 fibroblast lines.

( 9 )

 9 . Complex I was used as a loading control and was detected equally in both fibroblast lines. The decreased levels of SCO1 and COX proteins in SCO1 cells, therefore, validate SCO1 as a COXD cell line. Differential electrochemical signals are expected for COX activity in Control cells compared to SCO1-deficient fibroblast cells.

Fig. 2 .

 2 Fig. 2. Western blot results of mitoplasts isolated from Control and SCO1 fibroblast cells. (A) Fibroblasts fractionated by SDS-PAGE and blot were subject to antibodies against SCO1. VDAC1 was used as a loading control. Molecular weight (MW) protein ladder is indicated on the left. (B) Fibroblasts fractionated by 1D BN-PAGE and blot were subject to antibodies against complex IV. Complex I was used as a loading control.

Fig. 3 .

 3 Fig. 3. SECM 3D imaging of single living fibroblasts at 50 μm s -1 . Microelectrode polarized at +200 mV and -200 mV to scan Control (A and B) and SCO1 fibroblasts (C and D).

Fig. 4 .

 4 Fig. 4. SECM line scans across fibroblast cells. Imaging of single living Control (A and B) and SCO1 (C and D) fibroblasts in basic DMEM media containing 1 mM TMPD, with scan velocities ranging from 10 to 100 μm s -1 and the microelectrode biased at +200 V (A and C) and -200 V (B and D). Normalized currents shown (current divided by current in bulk solution). Insets show optical micrographs of the studied cells and the electrode edge path during line scan acquisition is represented by the black dashed lines.

  The average estimated k0 value obtained for COX activity from Control and SCO1 cells (n = 4 for each cell line) was 3.49 x10 -5 m s -1 and 2.10 x10 -5 m s -1 , respectively. These results indicate a statistically significantly slower COX kinetic rate for the COX-deficient cell line.Similarly, the average k0 value for CRE activity was determined to be equal to 4.16 x10 -5 m s -1 and 2.35 x10 -5 m s -1 for Control and SCO1 fibroblasts (n = 4 for each cell line), respectively.

Fig. 5 .

 5 Fig. 5. Summary of the numerical modeling results for the analysis of Control and SCO1 fibroblasts using SECM. (A, B) COX activity. (C-D) CRE activity. (A, C) The colored lines represent the simulated normalized peak currents for several substrate and sample kinetics ranging from 10 -8 to 10 1 m s -1 . The current signal for Control (square) is higher than the one for SCO1 fibroblast (circle). (B, D) The normalized current is taken at low Ps (<1) as a function of kinetic k0.

Acknowledgments

The authors SK, ST, and DL thank Drs. Christian Heinemann and Frank Wang from HEKA Elektronik GmbH for the technical support on the SPECM instrument and software integrations. The authors thank Dr. Eric Shoubridge (McGill University, Canada) for providing the SCO1 cells. SK acknowledges the University of Manitoba for seed funding through the University Research Grant Program (URGP). The Natural Sciences and Engineering Research Council of Canada (RGPIN-2019-05365) is acknowledged for its financial support.

funding, and wrote and revised the manuscript.

SECM measurements

All electrochemical experiments were performed using an ElProScan ELP3 system, with POTMASTER software (version V2 × 66) and an ElProScan Controller ESC 3 (HEKA Elektronik, Germany). A three-electrode setup consisting of an Ag/AgCl reference electrode, a 0.5 mm diameter platinum wire auxiliary electrode, and a 25 µm platinum microelectrode (RG = 2.8, HEKA Elektronik, Germany) was used. Prior to any measurement, the platinum microelectrode was polished using 0.05 µm alumina slurry (Buehler, USA) on an automated electrode polisher (LaboPol-20, Struers Inc., USA). The electrochemical behavior of TMPD in basic DMEM cell media (no FBS) was investigated using cyclic voltammetry, with measurements carried out at a potential scan rate of 100 mV s -1 .

All SECM imaging measurements were carried out in feedback mode. TMPD (1 mM) was used as a redox mediator and was dissolved in basic DMEM media (no FBS).

Prior to cell exposure, the TMPD solution was sterile-filtered using a 0.22 µm pore syringe filter (Millipore Sigma). Cells were incubated in the TMPD solution for 20 min at 37 ºC before SECM data acquisition. Target fibroblasts were identified using an optical microscope located underneath the SECM stage. Prior to commencing line scans, a tipto-substrate distance of 12 µm was achieved through a negative feedback approach curve over plastic in close proximity to a living cell of interest [START_REF] Kuss | High-Speed Scanning Electrochemical Microscopy Method for Substrate Kinetic Determination: Method and Theory[END_REF]. All biological samples for this study were scanned at constant height with scan velocities ranging from 10 to 100 μm s - 1 . During SECM data collection, cell dimensions, and morphology were constantly monitored using the SECM-integrated optical microscope. To ensure cell viability, SECM measurements were collected in under 30 minutes in the presence of the TMPD redox mediator.

Statistical analysis

All experiments were performed in quadruples (unless stated otherwise). Twosample t-Test was used to determine whether the difference in mean apparent heterogeneous rate constants between Control 65 and SCO1 fibroblast cells differed significantly. For both COX and CRE activity, a P < 0.10 was considered statistically significant. Statistical analyses were performed using Origin 2019.