Thi Phuong

Thuy Hoang

David Touboul
email: david.touboul@cnrs.fr

Comparison of internal energy distributions generated by supercritical fluid chromatography versus liquid chromatography hyphenated with electrospray high resolution mass spectrometry

Keywords: Internal energy distribution, thermometer ions, survival yield method, supercritical fluid chromatography, liquid chromatography, electrospray ionization

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Mass spectrometry (MS) is one of the most common analytical techniques employed in the field of chemical analysis. The internal energy (IE) distribution of ions in the gas phase have always attracted attention of MS specialists because this energy deposited into analytes during the ionization process directly affects the in-source fragmentation [START_REF] Drahos | Thermal energy distribution observed in electrospray ionization[END_REF][START_REF] Collette | Calibration of the internal energy distribution of ions produced by electrospray[END_REF][START_REF] Collette | Comparison of the internal energy distributions of ions produced by different electrospray sources[END_REF][START_REF] Vékey | Electron capture induced decomposition of the benzene C6H62+ ion[END_REF][START_REF] Gabelica | Influence of the capillary temperature and the source pressure on the internal energy distribution of electrosprayed ions[END_REF][START_REF] Williams | The internal energy distribution in fast atom bombardment/liquid secondary ionmass spectra[END_REF]. This classical phenomenon is usually not desired because it could lead to complicate the MS data interpretation and make quantification more complex [START_REF] Tu | Internal energy deposition in infrared matrix-assisted laser desorption electrospray ionization with and without the use of ice as a matrix[END_REF] even if in-source dissociation is required for structural analysis when tandem mass spectrometry is not available. Therefore, a control of the IE from the MS setting is crucial depending on the goal of the analyses such as study of non-covalent interactions [START_REF] Touboul | Ion internal energy distributions validate the charge residue model for small molecule ion formation by spray methods[END_REF], structural studies or construction of MS or MS/MS spectral libraries [START_REF] Gabelica | Internal energy and fragmentation of ions produced in electrospray sources[END_REF]. To evaluate the internal energy distribution, the 'thermometer molecules' method has been introduced by Wysocky et al. [START_REF] Wysocky | Internal energy distributions of isolated ions after activation by various methods[END_REF], together with the 'deconvolution method' [START_REF] Vékey | Electron capture induced decomposition of the benzene C6H62+ ion[END_REF] in the 80's. Since 2005, the 'survival yield' method described by De Pauw et al. [START_REF] Gabelica | Internal energy and fragmentation of ions produced in electrospray sources[END_REF] has been extensively employed for the determination of the internal energy distribution for soft ionization techniques [START_REF] Collette | Calibration of the internal energy distribution of ions produced by electrospray[END_REF][START_REF] Collette | Comparison of the internal energy distributions of ions produced by different electrospray sources[END_REF][START_REF] Tu | Internal energy deposition in infrared matrix-assisted laser desorption electrospray ionization with and without the use of ice as a matrix[END_REF][START_REF] Touboul | Ion internal energy distributions validate the charge residue model for small molecule ion formation by spray methods[END_REF][START_REF] Derwa | New basis for a method for the estimation of secondary ion internal energy distribution in "soft" ionisation techniques[END_REF][START_REF] Naban-Maillet | Internal energy distribution in electrospray ionization[END_REF][START_REF] Fu | Internal energy distribution of secondary ions under argon and bismuth cluster bombardments: "Soft" versus "Hard" desorption-ionization process[END_REF]. The benzylpyridinium salts considered as thermometer ions have been usually employed to probe the internal energy distribution using the survival yield method because they exhibit similar mass, structures, degrees of freedom as well as internal energy distribution and calculated dissociation energies are available [START_REF] Touboul | Ion internal energy distributions validate the charge residue model for small molecule ion formation by spray methods[END_REF]. Beside benzylpyridinium salts, some authors have introduced other classes of molecules to extend the validity of the technique such as protonated esters to study the influence of rearrangement reactions and protonated leucine encephalin to study the influence of molecular size by Naban-Maillet et al. [START_REF] Naban-Maillet | Internal energy distribution in electrospray ionization[END_REF], or other biomolecules by Flanigan IV et al. [START_REF] Flanigan | Determination of internal energy distributions of laser electrospray mass spectrometry using thermometer ions and other biomolecules[END_REF]. Understanding the influence of different parameters on the internal energy distribution appears helpful in selecting the suitable setting parameters for each type of analysis. Many articles were dedicated to the comparison of the internal energy distribution generated by different well-recognized soft ionization techniques such as electrospray (ESI) [START_REF] Gabelica | Influence of the capillary temperature and the source pressure on the internal energy distribution of electrosprayed ions[END_REF][START_REF] Touboul | Ion internal energy distributions validate the charge residue model for small molecule ion formation by spray methods[END_REF][START_REF] Naban-Maillet | Internal energy distribution in electrospray ionization[END_REF][START_REF] Harris | Comparison of the internal energy deposition of direct analysis in real time and electrospray ionization time-of-flight mass spectrometry[END_REF], desorption electrospray ionization DESI [START_REF] Nefliu | Internal energy distributions in desorption electrospray ionization (DESI)[END_REF][START_REF] Badu-Tawiah | Non-aqueous spray solvents and solubility effects in desorption electrospray ionization[END_REF], laser ablation electrospray ionization [START_REF] Nemes | Internal energy deposition and ion fragmentation in atmosphericpressure mid-infrared laser ablation electrospray ionization[END_REF], matrix-assisted laser desorption ionization MALDI [START_REF] Tu | Internal energy deposition in infrared matrix-assisted laser desorption electrospray ionization with and without the use of ice as a matrix[END_REF][START_REF] Fu | Internal energy distribution of secondary ions under argon and bismuth cluster bombardments: "Soft" versus "Hard" desorption-ionization process[END_REF][START_REF] Flanigan | Determination of internal energy distributions of laser electrospray mass spectrometry using thermometer ions and other biomolecules[END_REF][START_REF] Nemes | Internal energy deposition and ion fragmentation in atmosphericpressure mid-infrared laser ablation electrospray ionization[END_REF][START_REF] Luo | Internal energy of ions generated by matrix-assisted laser desorption/ionization[END_REF][START_REF] Gabelica | Internal energy build-up in matrix assisted laser desorption/ionization[END_REF], direct analysis in real time DART [START_REF] Harris | Comparison of the internal energy deposition of direct analysis in real time and electrospray ionization time-of-flight mass spectrometry[END_REF], or even fast atom bombardment FAB [START_REF] Derwa | New basis for a method for the estimation of secondary ion internal energy distribution in "soft" ionisation techniques[END_REF][START_REF] De Pauw | Internal energy distribution of ions emitted in secondary ion mass spectrometry[END_REF]. It was shown that the internal energy distribution depends on 1) the minimal energy necessary to release the analytes from its matrix/solution and 2) the collisions of the molecular ion occurring in the source [START_REF] Gabelica | Internal energy and fragmentation of ions produced in electrospray sources[END_REF]. Several instrumental parameters play major role in the distribution of the internal energy such as cone and skimmer voltages [START_REF] Collette | Comparison of the internal energy distributions of ions produced by different electrospray sources[END_REF][START_REF] Touboul | Ion internal energy distributions validate the charge residue model for small molecule ion formation by spray methods[END_REF][START_REF] Voyksner | Investigation of collisional-activation decomposition process and spectra in the transport region of an electrospray single quadrupole mass spectrometer[END_REF], capillary temperature and voltage [START_REF] Gabelica | Influence of the capillary temperature and the source pressure on the internal energy distribution of electrosprayed ions[END_REF], collision energy [START_REF] Touboul | Ion internal energy distributions validate the charge residue model for small molecule ion formation by spray methods[END_REF][START_REF] Naban-Maillet | Internal energy distribution in electrospray ionization[END_REF], the nature [START_REF] Collette | Comparison of the internal energy distributions of ions produced by different electrospray sources[END_REF][START_REF] Douglas | Mechanism of the collision-induced dissociation of polyatomic ions studied by triple quadrupole mass spectrometry[END_REF][START_REF] Schmidt | Influence of pressure in the first pumping stage on analyte desolvation and fragmenation in nano-ESI MS[END_REF] and the pressure [START_REF] Schmidt | Influence of pressure in the first pumping stage on analyte desolvation and fragmenation in nano-ESI MS[END_REF] of the collision gas, gas flow rate and temperature [START_REF] Harris | Comparison of the internal energy deposition of direct analysis in real time and electrospray ionization time-of-flight mass spectrometry[END_REF], but some other parameters did not show any influence as the nebulizing gas pressure and flow rate, the distance between the emitter and the inlet of the mass spectrometer [START_REF] Touboul | Ion internal energy distributions validate the charge residue model for small molecule ion formation by spray methods[END_REF]. Recent researches have paid attention to the sample state which has been shown to strongly affect the internal energy distribution. Flanigan IV and coworkers have demonstrated that analysis of solid samples by laser electrospray mass spectrometry LEMS resulted in more energy deposition into the thermometer ions than liquid samples [START_REF] Flanigan | Determination of internal energy distributions of laser electrospray mass spectrometry using thermometer ions and other biomolecules[END_REF].

In recent years, supercritical fluid chromatography SFC using supercritical CO2 as one of the solvents for the mobile phase has attracted more and more attention [START_REF] West | Current trends in supercritical fluid chromatography[END_REF][START_REF] Liu | The application of supercritical fluid chromatography in food quality and food safety: An overview[END_REF]. This technique is considered as a green analytical technique with various interests such as short analysis times, high efficiency, stability for samples, and safety for users [START_REF] Méjean | Quantification of tocopherols and tocotrienols in soybean oil by supercritical-fluid chromatography coupled to high-resolution mass spectrometry[END_REF][START_REF] Hoang | Annotation and quantification of N-acyl homoserine lactones implied in bacterial quorum sensing by supercritical-fluid chromatography coupled with high-resolution mass spectrometry[END_REF][START_REF] Schulze | A rapid method for quantification of persistent and mobile organic substances in water using supercritical fluid chromatography coupled to high-resolution mass spectrometry[END_REF][START_REF] Akbal | Supercritical fluid chromatography-mass spectrometry using data independent acquisition for the analysis of polar metabolites in human urine[END_REF]. As far as we know, the internal energy distributions of ions formed after this separation method coupled to ESI has never been examined.

The main objective of the present work was to investigate and compare the internal energy distributions of ions generated by SFC and liquid chromatography (LC) coupled with ESI source fitted on a quadrupole time-of-flight QTOF mass spectrometer. Influence of chromatographic parameters, such as composition and flow rate of the mobile phase, and MS source parameters were examined for SFC and LC to enlighten different ionization conditions.

Material and methods

Chemical and reagents

Carbon dioxide (purity 99.7% or greater) was purchased from Air Liquide (Grigny, France). Methanol (HPLC grade) was purchased from J.T. Baker (Center Valley, PA, USA). Absolute ethanol (HPLC isocratic grade) was purchased from Carlo Erba (Val de Reuil, France). 2-propanol (LC-MS grade) was purchased from Fisher Scientific (Illkirch, France).

Benzylpyridinium salts (Table 1) were kindly provided by Prof. D. Rondeau (Université de Bretagne Occidentale, Brest, France) except for the p-cyanobenzylpyridinium salt which was purchased from Otava Chemicals Ltd. (Kyiv, Ukraine). The stock solutions of each salt were separately prepared in MeOH/H2O

(1/1, v/v) at a concentration of 0.2 mg/mL. The solutions were kept at -20 °C. Two mixtures were prepared prior to analyses. The first mixture (Mix-01) consisted of four benzylpyridinium salts including p-OCH3, p-CH3, p-Cl, p-F, and the second one (Mix-02) consisted of p-CN, p-NO2, m-OCH3 and m-CH3.

Analytical conditions 2.2.1. Supercritical fluid chromatography (SFC) analysis

SFC analyses were performed using a 1260 Infinity analytical SFC system (Agilent Technologies, Waldbronn, Germany). This system was composed of a controller module (G1170A), a SFC module (G4301A), a binary pump (G4302A), a degasser (G4225A), an auto sampler (G4303A) which was thermostated (kept at 5 °C by a thermostat G1130B) and equipped with a 1.2 µL injection loop, a thermostatically controlled column compartment (G1316C), a UV-DAD detector (G1315C), a back pressure regulator BPR (G4301A), a make-up isocratic pump (G1310B). The isocratic pump allows to deliver a make-up solvent, generally it is a polar solvent such as alcohol or acetonitrile with or without additive, in the mobile phase after the UV-DAD in order to enhance the solubility of analytes which could be precipitated due to CO2 decompression. In our SFC system, a T-union was placed in front of the BPR in order to split the mobile phase into two fractions, one fraction to BPR and the other one to MS. When coupling with a mass spectrometer, a caloratherm preheater (Sandra Selerity Technologies, Kortrijk, Belgium) set at 60 °C was installed at the entrance of the ion source to avoid the freezing of the mobile phase.

The mobile phase consisted of CO2 (solvent A) and co-solvent (solvent B). The bypass mode, i.e. no use of chromatographic column, was employed. The flow rate was fixed at 1 mL/min and the run time lasted 3 min in isocratic mode. Different SFC conditions were studied such as the percentage and the nature of co-solvent, pressure at back-pressure regulator (BPR). The generic SFC method was set as follows: mobile phase containing 95% CO2 and 5% MeOH with flow rate of 1 mL/min, BPR set at 90 bar and 60 °C.

Liquid chromatography (LC) analysis

LC analyses were carried out on a 1260 Infinity analytical HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with a capillary pump (G1376A), a flexible pump (G7104C), a degasser (G1379), an auto sampler (G7129C) which was thermostated (kept at 5 °C) and a thermostatically controlled column compartment (G7116A). Injection volume was set at 5 µL. The mobile phase consisted of water (solvent A) and methanol (solvent B). The bypass mode was employed. The run time was fixed at 3 min in isocratic mode. LC conditions including the percentage of methanol and the flow rate of the mobile phase were studied. The generic LC method was set as follows: mobile phase containing MeOH/H2O 50:50 (v/v) with flow rate of 0.3 mL/min.

Mass spectrometry (MS) conditions

The SFC and LC systems were coupled with a 6540 quadrupole time-of-flight mass spectrometer (QToF 6540, Agilent Technologies, Massy, France). A conventional electrospray (ESI) source was employed.

After the ESI source, a heated nitrogen flow called drying gas is employed to eliminate the micro-drops of solvents which still present after the ESI source. The ions formed will then enter a capillary covered with platinum at each end, allowing the application of a voltage called Vcap. At the outlet of the capillary, analytes will be subjected to a voltage called fragmentor voltage. Influence of drying gas temperature, the capillary voltage and the fragmentor voltage on the internal energy distribution were studied. Analyses were carried out with following parameters: positive ionization mode, drying gas flow rate 10 L/min, nebulizer pressure 50 psi, skimmer voltage 45 V, and Octopole 1 RF Voltage 750 V. For the generic SFC and LC methods, MS parameters were set as follow: drying gas temperature 350 °C, Vcap 3500 V, fragmentor 160 V. Full-scan mode was employed in a range of m/z 50-1700 at 2 GHz, giving a mass resolution higher than 25 000 at m/z 922. Calibration solution containing two internal reference compounds, purine C5H4N4 m/z 121.0509 and hexakis (1H, 1H, 3H-tetrafluoropentoxy) phoshazene HP-921 C18H18O6N3P3F24 m/z 922.0098, was continuously introduced resulting in mass accuracy below 5 ppm. Recorded data were analyzed with MassHunter Workstation B.08.00. The OriginPro 2015 (OriginLab Corporation, Northampton, MA USA) software was used to plot a scatter graph for the experimental data, to fit the sigmoidal curves using Boltzmann function, to differentiate the sigmoidal curves to get the internal energy distributions and to calculate the mean internal energy.

Results and discussion

Determination of the internal energy distribution

The internal energy distributions of thermometer ions were calculated based on the survival yield (SY) method described by Gabelica et al. [START_REF] Gabelica | Fast gas-phase hydrogen/deuterium exchange observed for a DNA G-quadruplex[END_REF]. The SY method assumes that all molecular ions having an internal energy higher than the critical energy (E0) will dissociate into fragment ions, and those having an internal energy lower than E0 will not dissociate and remain intact. The SY of a molecular ion is defined by the following equation:

= M+ M+ + F+
where IM+ is the intensity of the molecular ion and IF+ is the intensity of the fragment ion.

The SY are then plotted as a function of the critical energy E0. Assuming that no molecular ion remains (SY=0) when the critical energy is zero and there is no fragment ion (SY=1) when the critical energy is 4 eV, two data points (E0=0 eV; SY=0 and E0=4 eV; SY=1) are added to the SY curve to approximate low and high critical energy thresholds. The internal energy distribution was then calculated based on the derivative dSY(E)/d(E) [START_REF] Gabelica | Fast gas-phase hydrogen/deuterium exchange observed for a DNA G-quadruplex[END_REF]. The SY(E) curve was first fitted by a sigmoidal curve using a Boltzmann function because the internal energy distribution generated by an electrospray source can be assumed to follow a thermal-like Boltzman distribution [START_REF] Turecek | Temperature effects in mass spectrometry. Estimation of thermal energies of organic molecules[END_REF][START_REF] Drahos | Determination of the thermal energy and its distribution in peptides[END_REF][START_REF] Hoxha | Mechanism of collisional heating in electrospray mass spectrometry: ion trajectory calculations[END_REF].

The internal energy distribution was then obtained by differentiating the sigmoidal curve. Its Gaussianlike shape allows to confirm the previous approximation. For internal energy study, benzylpyridinium salts are widely utilized and considered as thermometer ions [START_REF] Collette | Comparison of the internal energy distributions of ions produced by different electrospray sources[END_REF][START_REF] Derwa | New basis for a method for the estimation of secondary ion internal energy distribution in "soft" ionisation techniques[END_REF][START_REF] De Pauw | Internal energy distribution of ions emitted in secondary ion mass spectrometry[END_REF]. These molecules present a common fragmentation pattern with a cleavage of the C-N bond resulting in a benzyl cation and a pyridine neutral (Figure 1) [START_REF] Touboul | Ion internal energy distributions validate the charge residue model for small molecule ion formation by spray methods[END_REF].

In this study, eight substituted (para-and meta-) benzylpyridinium salts were employed to evaluate the internal energy distributions of ions generated by electrospray after liquid or supercritical fluid chromatography (Table 1). There are many calculation approaches used to detect the critical energy (E0) depending on the level of calculation [START_REF] Gabelica | Internal energy and fragmentation of ions produced in electrospray sources[END_REF][START_REF] Debord | Measuring the internal energies of species emitted from hypervelocity nanoprojectile impacts on surfaces using recalibrated benzylpyridinium probe ions[END_REF]. In this study, we used the values presented by Gabelica et al. [START_REF] Gabelica | Internal energy and fragmentation of ions produced in electrospray sources[END_REF] because their critical energies matched well with experimental survival yields. It is important to note that the obtained energies were not the absolute values of the internal energy because the kinetic shift was not taken into account in our study but it allowed to indicate relative changes related to chromatographic or/and source parameters. The survival yields and the internal energy distributions of benzylpyridinium salts using the generic SFC-MS condition was shown in Figure 2. The R-squared value was 0.9932 indicating that the Boltzmann function fitted well with the data.

Influence of SFC-ESI-MS conditions on the internal energy distributions of thermometer ions

In order to better understand the quantity of energy deposited on thermometer ions during SFC-ESI-MS analysis, different experimental conditions were explored. First, the percentage of co-solvent methanol (MeOH) in the mobile phase was varied from 0 to 40%, that is typical of SFC gradient. No significant difference was observed on the internal energy distribution (Figure 3-a). The influence of the chemical nature of the co-solvent, i.e. MeOH, MeOH with 20 mM of ammonium acetate (AcONH4), mixture of MeOH/EtOH (1/1, v/v), EtOH and IPA, was then studied at fixed value of 5%. The results showed that this parameter did not influence the internal energy distributions of thermometer ions (Figure 3-b). In the SFC employed, the principal constituent of the mobile phase is CO2 (95%) and the mobile phase is always kept in supercritical state. The CO2 is decompressed at the MS entrance leading to rapid evaporation.

Moreover, co-solvents are evaporated easily at the MS entrance when added at 5% in supercritical CO2.

Thus no change in the internal energy of ions was observed when varying the composition of the mobile phase in SFC. The effect of pressure of the back pressure regulator (BPR) was also evaluated at different values, i.e. 90, 110, 130 and 150 bar, leading to no effect on the internal energy distribution (Figure 3-c).

Different flow rates of mobile phase and make-up solvent were studied leading to no influence of these parameters on the survival energy distribution of thermometer ions (Figures 3-d and3-e).

The impact of the MS instrumental parameters, including the drying gas temperature, the fragmentor voltage (Frag) and the capillary voltage (Vcap), was then considered. The drying gas temperature and the fragmentor voltage were found to affect the internal energy distribution whereas the Vcap exhibited no influence (Figures 3-f, 3-g and 3-h). The drying gas temperature allows the desolvation of the charged droplets and/or ions out of the nebulizer. When increasing the temperature, thermal ion activation is occurring. The kinetic energy as well as the velocity of the molecules is greater, conducting to the increase of the internal energy and the decrease of the SY observed in this study. This result is in accordance with previous studies related to ESI [15]. This trend was more pronounced for the fragmentor voltage. This voltage applied between the capillary and the skimmer could induce the ion fragmentation after collisions with the remaining gas at the interface. At a fragmentor voltage of 120 V, all thermometer ions were stable with SY close to 98% except for the most unstable ion, p-OCH3, with SY of 71%. When the fragmentor voltage was increased up to 180 V, almost all precursor ions were fragmented with the SY around 20%, except for p-OCH3, p-CN and p-NO2 with the SY of 4%, 52% and 88%, respectively.

Influence of LC-ESI-MS conditions on the internal energy distribution of thermometer ions

In order to compare the influence of the eluent state (supercritical and liquid) on the internal energy distribution of thermometer ions, the study for LC-ESI-MS was performed on the same mass spectrometer using the same MS conditions as in SFC analyses. The drying gas temperature at 350 °C and the fragmentor voltage at 160 V were selected for the generic method because the SY values were well distributed along the sigmoid fit (Figure 2). The LC mobile phase consisted of water and methanol. The influence of the percentage of methanol in the mobile phase as well as the flow rate were first examined.

No influence was observed on the internal energy distribution (Figure 4) indicating that the internal energy distributions of thermometer ions was independent of the nature and the flow rate of the mobile phase. Three MS parameters previously studied for SFC were also evaluated for LC, i.e. the drying gas temperature, the fragmentor voltage and the capillary voltage. We observed the same influence trend of these MS parameters for LC as for SFC: the drying gas temperature and the fragmentor voltage determined the internal energy distributions of thermometer ions, but the capillary voltage had no significant effect.

Comparison of the internal energy distributions formed after SFC-and LC-ESI-MS

The internal energy distributions of thermometer ions formed by SFC-ESI-MS was compared with that obtained by LC-ESI-MS on the same MS system with the same parameters (Figure 5). The molecule having higher E0 is more stable, implying higher SY.. The order of two meta-substituted salts (m-CH3 and m-OCH3) was inversed between SFC and LC. However, their survival yields were very close in all studied conditions.

The comparison of the survival yield of thermometer ions formed by SFC-and LC-ESI-MS method using the generic condition was introduced in Figure 6. In the case of SFC, the thermometer ion has lower survival yield than that in LC when using the same MS condition. This trend was well observed for p-CH3, p-Cl, p-F, m-CH3, m-OCH3 but was less pronounced for m-OCH3, p-CN and p-NO2. These results suggested that the influence of the chromatographic method on the survival yield was compounddependent.

The comparison of the mean internal energies was shown in Figure 7. For the same analytical conditions, SFC-ESI-MS systemically led to higher mean internal energies than the LC-ESI-MS whatever the MS source parameters. This clearly indicates that SFC-ESI-MS allows to deposit more internal energy into ions in the gas phase than the LC-ESI-MS. The variation of the mean internal energies with the drying gas temperature and the fragmentor voltage were all linear in accordance with the literature [START_REF] Drahos | Determination of the thermal energy and its distribution in peptides[END_REF][START_REF] Vékey | Internal energy effects in mass spectrometry[END_REF][START_REF] Schneider | Collision-induced dissociation of ions within the orifice-skimmer region of an electrospray mass spectrometer[END_REF].

Furthermore, the curves of the internal energy distributions for SFC and LC using the same MS parameters (Figure 8) were very close implying that the ion formation mechanism was similar, known as the charge evaporation model [START_REF] Touboul | Ion internal energy distributions validate the charge residue model for small molecule ion formation by spray methods[END_REF][START_REF] Dole | Molecular beams of macroions[END_REF]. The CO2 in SFC has been described to enhance the ESI nebulizing process by promoting the desolvation of the analytes [START_REF] Akbal | Hyphenation of packed column supercritical fluid chromatography with mass spectrometry: where are we and what are the remaining challenges?[END_REF]. Consequently, a rapid desolvation leads to colder ions and a softer ionization process. However, the experimental data in this study was not in accordance with this hypothesis because the SYs of thermometer ions obtained by SFC-ESI-MS were all lower than that obtained by LC-ESI-MS. There are two hypotheses which could explain this observation.

The first one concerns rapid CO2 decompression after the down-stream T-union at the MS inlet. CO2 rapidly evaporates leading to smaller droplets or aggregates as well as smaller droplet surface tension comparing to LC-ESI-MS. All thermal activation at the MS interface will therefore be deposited on the analyte resulting in more fragmentation in SFC than in LC in the same MS conditions. The second one relates the high percentage of CO2 in the nebulizing gaz. In SFC, the transformation from supercritical to atmospheric conditions occuring at the MS inlet has been shown to cause a 450-fold expansion [START_REF] Gabelica | Internal energy and fragmentation of ions produced in electrospray sources[END_REF]. At our analysis conditions, flow rate of the supercritical mobile phase was fixed at 1 mL/min equivalent to about 0.45 L/min after expansion whereas the N2 nebulizing gas was kept at 10 L/min, leading to a percentage of CO2 in nebulizing gas about 5% compared to 100% N2 for LC. It has been showed that the nature of the nebulizing gas influences the internal energy distribution [START_REF] Collette | Calibration of the internal energy distribution of ions produced by electrospray[END_REF][START_REF] Gabelica | Internal energy and fragmentation of ions produced in electrospray sources[END_REF]. Adding CO2 in nebulizing gas could shift the internal energy distribution to higher value in comparison with pure N2 as showed in the study of Collette et al. [START_REF] Collette | Calibration of the internal energy distribution of ions produced by electrospray[END_REF]. This could be explained by the direct relationship between the center of mass energy with the mass of the nebulizing gas [START_REF] Schneider | Collision-induced dissociation of ions within the orifice-skimmer region of an electrospray mass spectrometer[END_REF].

Conclusions

Our study demonstrated that the chromatographic parameters (the nature, the composition, the flow rate of the mobile phase, the pressure of the BPR) played a minor role in the internal energy distribution of the ions after ESI, either in SFC or in LC. The internal energy distribution mainly depended on the drying gas temperature and the fragmentor voltage but it was independent of the capillary voltage. By comparing the internal energy distributions formed by SFC-and LC-ESI-MS, we observed that analysis by SFC resulting from more fragmentation and higher internal energy than LC analysis resulting to a more efficient desolvation process before the entrance of the mass spectrometer. Therefore, when method transfer from SFC-MS to LC-MS, or vice-versa, is performed, MS parameters must be finely tuned to optimize the quantity of intact ions required for high resolution mass spectrometry detection or even for single/multiple reaction monitoring experiments.

Figure captions

Fig. 1 .

 1 Figure captionsFig. 1. MS/MS fragmentation pattern of benzylpyridinium cations into the benzylium cation and neutral pyridine.

Fig. 2 .

 2 Fig. 2. (a) Survival yields of the benzylpyridinium ions plotted as a function of their measured critical energies for SFC-ESI-MS using the generic method: Mobile phase was composed of CO2/MeOH 95/5,

Fig. 3 .

 3 Fig. 3. Internal energy distributions of thermometer ions for SFC-ESI-MS in different studied conditions: (a) Influence of the percentage of MeOH as co-solvent in the mobile phase on the internal energy distribution. (b) Influence of the nature of the co-solvent in the mobile phase on the internal energy distribution. (c) Influence of the pressure of the back pressure regulator (BPR) on the internal energy distribution. (d) Influence of the flow rate of the mobile phase on the internal energy distribution. (e) Influence of the flow rate of the make-up solvent on the internal energy distribution. (f) Influence of the drying gas temperature on the internal energy distribution. (g) Influence of the fragmentor voltage on the internal energy distribution. (h) Influence of the capillary voltage on the internal energy distribution. For analyses in (a), (b), (c), (d), (e): Drying gas temperature at 350 °C, voltage of the capillary and of the fragmentor at 3500 V and 160 V respectively. For analyses in (f), (g), (h): Mobile phase was composed of CO2/MeOH 95/5, back-pressure regulator (BPR) at 90 bar and 60 °C.

Fig. 4 .

 4 Fig. 4. Internal energy distributions of thermometer ions for LC-ESI-MS in different studied conditions: (a) Influence of the percentage of MeOH in the mobile phase on the internal energy distribution. (b) Influence of the flow rate of the mobile phase on the internal energy distribution. (c) Influence of the drying gas temperature on the internal energy distribution. (d) Influence of the fragmentor voltage on the internal energy distribution. (e) Influence of the capillary voltage on the internal energy distribution. For analyses in (a), (b): Drying gas temperature at 350 °C, voltage of the capillary and of the fragmentor at 3500 V and 160 V respectively. For analyses in (c), (d), (e): Mobile phase was composed of MeOH/H2O 50/50 v/v, flow rate at 0.3 mL/min.

Fig. 5 .

 5 Fig. 5. Breakdown curves for the benzylpyridinium ions as a function of the drying gas temperature (SFC-1 and LC-1), the fragmentor voltage (SFC-2 and LC-2) and capillary voltage (SFC-3 and LC-3).

Fig. 6 .

 6 Fig. 6. Comparison of the survival yield of thermometer ions analyzed by SFC-and LC-ESI-MS in the generic method.

Fig. 7 .

 7 Fig. 7. Comparison of the mean survival yield of thermometer ions analyzed by SFC-and LC-ESI-MS in different (a) drying gas temperatures (b) fragmentor voltages and (c) capillary voltages.

Fig. 8 .

 8 Fig.8. Comparison of the internal energy distribution for SFC (red curve) and LC (black curve) using common generic ESI-MS parameters.

Table 1 .

 1 The benzylpyridinium ions used for the study of the internal energy distribution.

	Solution R a	m/z (M + ) b m/z (F + ) c E0 (eV) d
		p-OCH3	200.1070	121.0653 1.508
		p-CH3	184.1121	105.0704 1.767
	Mix-01
		p-Cl	204.0575	125.0158 1.899
		p-F	188.0870	109.0454 1.867
		p-CN	195.0917	116.0500 2.097
	Mix-02	p-NO2	215.0815	136.0399 2.352
		m-CH3	184.1121	105.0704 1.901
		m-OCH3 200.1070	121.0653 1.945

a Substituent group of the benzylpyridinium ions b M + : Molecular ion c F + : Fragment ion d E0 (eV): Critical energy calculated at the semi-empirical Austin Method 1 (AM1)

[START_REF] Gabelica | Internal energy and fragmentation of ions produced in electrospray sources[END_REF]

Acknowledgements

This work was supported by the Agence Nationale de la Recherche (Grant ANR-16-CE29-0002-01 CAP-SFC-MS) and a grant from Région Ile-de-France (DIM Analytics).

Declaration of competing interest

The authors declare that they have no conflict of interest.


Jade Aurrière

David Goudenège

Olivier R Baris

Magalie Boguenet

Pascale May-Panloup

Guy Lenaers

Salim Khiati
email: salim.khiati@univ-angers.fr

Cancer/Testis Antigens into mitochondria: a hub between spermatogenesis, tumorigenesis and mitochondrial physiology adaptation

Keywords: Mitochondrion, Cancer/Testis Antigen, localization, spermatogenesis, tumorigenesis

Cancer/Testis Antigens (CTAs) genes are expressed only during spermatogenesis and tumorigenesis.

Both processes share common specific metabolic adaptation related to energy supply, with a glucose to lactate gradient, leading to changes in mitochondrial physiology paralleling CTAs expression. In this review, we address the role of CTAs in mitochondria (mitoCTAs), by reviewing all published data, and assessing the putative localization of CTAs by screening for the presence of a mitochondrial targeting sequence (MTS). We evidenced that among the 276 CTAs, five were already shown to interfere with mitochondrial activities and 67 display a potential MTS.

I. INTRODUCTION -DISCOVERING CTAS

Cancer/Testis Antigens (CTA) represent a group of proteins whose expression under physiological conditions is restricted to testis, but also activated in many human cancers. The first CTA, MZ2-E, named MAGE-A1, was identified in 1991 as a melanoma tumor antigen, recognized by cytotoxic T lymphocytes in vitro [START_REF] Van Der Bruggen | A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma[END_REF]. Later, the screening of a recombinant cDNA expression library, built from sera of Melanoma patients, unraveled a new family of antigens sharing two features [START_REF] Carrero-Valenzuela | A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening[END_REF]: i) mRNA expression restricted to testis, and eventually to very few other tissues, and ii) mRNA expression activated during tumor development.

With the discovery of a larger number of CTAs, their molecular characteristics became more detailed :

i) many CTAs are localized on the X chromosome, where they are organized in multigenic "operonlike" clusters, ii) their expression can be correlated with tumor growth and stage, and iii) they can be activated by hypomethylation and inhibition of histone deacetylation in vitro [START_REF] Scanlan | Cancer/testis antigens: an expanding family of targets for cancer immunotherapy[END_REF].

As of today, 276 CTAs have been identified and compiled in a unique CTA-Database (http://www.cta.lncc.br/) [START_REF] Almeida | CTdatabase: a knowledge-base of high-throughput and curated data on cancer-testis antigens[END_REF].

With their expansion, new classification criteria emerged. First, CTAs were divided into two groups based on their chromosomal localization: X-CTAs, localized on the X-chromosome, and non-X CTAs, localized on autosomal chromosomes. More than half of the CTAs belong to the first group and are organized in multigene families, while non-X CTAs genes are dispersed along the 22 autosomes.

Second, although CTAs are testis-restricted expression, some of them are expressed under physiological conditions in few other tissues, mainly in the brain or in ovary. This led to a further classification in three groups: testis-restricted, testis/brain-restricted and testis-selective, depending on their expression pattern [START_REF] Hofmann | Genome-wide analysis of cancer/testis gene expression[END_REF].

II. CTAS EXPRESSION DURING CANCER

CTAs expression can be specific to a single tumorigenesis step or common to all steps of cancer development, leading to three expression profiles. First, most CTAs are broadly expressed during metastasis, while less frequently in primary tumors. For example, MAGE-A1 expression is observed in 51% of distant metastasis, but only in 20% of melanoma primary tumors. Similarly, MAGE-A4 is expressed in 44% of metastatic malignant melanoma, but only in 9% of primary tumors [START_REF] Barrow | Tumor Antigen Expression in Melanoma Varies According to Antigen and Stage[END_REF]. These features suggest that CTAs are biomarkers of poor prognosis in patients with cancer.

Second, co-expression of different CTAs in one cancer type is often observed, notably for X-CTAs.

For example, in Head and Neck Squamous Cell Carcinoma (HNSCC), the simultaneous expression of five CTAs (SPANX-C, D, MAGEB2, MAGEA1, MAGEB6 and CXORF48) was reported in 27% to 71.9% of analyzed HNSCC samples, depending of the malignant phenotype and the prognosis [START_REF] Zamunér | A Comprehensive Expression Analysis of Cancer Testis Antigens in Head and Neck Squamous Cell Carcinoma Revels MAGEA3/6 as a Marker for Recurrence[END_REF]. Finally, it was observed that co-expression of multiple CTAs at later stages of cancer worsens the prognosis. In this respect, multiple myeloma patients expressing more than 6 CTAs have a much poor prognosis than those expressing less than 6 CTAs [START_REF] Andrade | Prognostic impact of cancer/testis antigen expression in advanced stage multiple myeloma patients[END_REF]. This can be related to processes allowing cancer cells to recombine their genome, evade apoptosis and favor tissue invasion [START_REF] Hanahan | The Hallmarks of Cancer[END_REF]. Indeed, co-expression of FMR1NB, NXF2, and SSX2, induces genomic instability, while expression of SSX and CAGE sustains tumor growth. In addition, the expression of MAGE-A, MAGE-B, MAGE-C, GAGE, PAGE4 and CAGE inhibits apoptosis, while the expression of MAGE-C2, GAGE, XAGE1, CAGE and CT45A1 induces cancer invasion and metastasis [START_REF] Gjerstorff | Oncogenic cancer/testis antigens: prime candidates for immunotherapy[END_REF]. Owing to their unique expression profiles, CTAs are privileged targets for the development of immunotherapy cancer treatments. This led to the development of MAGE family members and NY-ESO1 antibody-targeted clinical trials [START_REF] Cheng | Cancer/testis (CT) antigens, carcinogenesis and spermatogenesis[END_REF][START_REF] Thomas | NY-ESO-1 Based Immunotherapy of Cancer: Current Perspectives[END_REF][START_REF] Vansteenkiste | Efficacy of the MAGE-A3 cancer immunotherapeutic as adjuvant therapy in patients with resected MAGE-A3-positive non-small-cell lung cancer (MAGRIT): a randomised, double-blind, placebo-controlled, phase 3 trial[END_REF]. Nevertheless, a better understanding of CTAs' functions in tumorigenesis is required in order to identify other relevant targets for new anti-cancer therapies.

III. CTAS EXPRESSION DURING SPERMATOGENESIS

Spermatogenesis occurs in testis in seminiferous tubules, to produce mature functional spermatozoa [START_REF] Neto | Spermatogenesis in humans and its affecting factors[END_REF]. The seminiferous tubule is composed of somatic cells and Sertoli cells supporting the development of the germline cells: spermatogonia, spermatocytes, spermatids and spermatozoa (Figure 1). Spermatogonia germ cells, which are located at the periphery of the seminiferous tubules, are in contact with blood. They proliferate and differentiate into primary spermatocytes by mitotic division. Primary spermatocytes undergo meiosis leading respectively to secondary spermatocytes and round spermatids, which are haploid cells. Round spermatids progressively engage in a differentiation phase leading to the development of mature spermatozoa. These processes are supported by the Sertoli cells, which are essential for germ cell line development, notably by producing and providing energy.

The volume of these cells is consistently important so that they can support simultaneously about 30 germ cells at various developmental stages [START_REF] Russell | A comparative study in twelve mammalian species of volume densities, volumes, and numerical densities of selected testis components, emphasizing those related to the Sertoli cell[END_REF] (Figure 1).

Although the functions of CTAs during spermatogenesis are poorly understood, their expression patterns have been well documented. Some CTAs are germ cell specific, whereas others are expressed during meiosis or at post-meiotic stages. Similar to the tumorigenesis process, some CTAs are expressed only during a single specific spermatogenesis step, while others are expressed during many steps. Only a few CTAs are expressed during all spermatogenesis steps, such as the Magphinin family [START_REF] Cheng | Cancer/testis (CT) antigens, carcinogenesis and spermatogenesis[END_REF]. Notably, X-CTAs are highly expressed during the first stages of spermatogenesis, before meiosis. In addition, NY-ESO-1, MAGEA1 and SSX family members are strongly expressed in spermatogonia, in mitotically proliferating germ cells and in primary spermatocytes [START_REF] Satie | The Cancer-Testis Gene, NY-ESO-1, Is Expressed in Normal Fetal and Adult Testes and in Spermatocytic Seminomas and Testicular Carcinoma In Situ[END_REF][START_REF] Stoop | Reactivity of Germ Cell Maturation Stage-Specific Markers in Spermatocytic Seminoma: Diagnostic and Etiological Implications[END_REF][START_REF] Takahashi | Identification of MAGE-1 and MAGE-4 Proteins in Spermatogonia and Primary Spermatocytes of Testis 6. The Gene Ontology Resource: 20 years and still GOing strong[END_REF]. Conversely, non-X CTAs, such as ACRBP (also known as OY-TES-1) and SPA17, are mostly expressed in post-meiotic phases, from the round spermatids step to the maturation of spermatozoa [START_REF] Cheng | Cancer/testis (CT) antigens, carcinogenesis and spermatogenesis[END_REF].

Importantly, expression of many CTAs is essential for spermatogenesis, as knocking out their genes in mice (Knockout, KO) results in infertility and altered sperm cells [START_REF] Babatunde | Cancer/Testis genes in relation to sperm biology and function[END_REF]. For example, PIWIL2 and TDRD1 KO male mice are infertile, with early meiotic prophase arrest during spermatogenesis [START_REF] Kuramochi-Miyagawa | Mili, a mammalian member of piwi family gene, is essential for spermatogenesis[END_REF][START_REF] Reuter | Loss of the Mili-interacting Tudor domain-containing protein-1 activates transposons and alters the Mili-associated small RNA profile[END_REF], while ADAM2 and ROPN1 KO male mice exhibit compromised morphology or migration of sperm cells [START_REF] Fiedler | Loss of R2D2 Proteins ROPN1 and ROPN1L Causes Defects in Murine Sperm Motility, Phosphorylation, and Fibrous Sheath Integrity1[END_REF][START_REF] Han | Impaired sperm aggregation in Adam2 and Adam3 null mice[END_REF]. Moreover, although fertile, THEG and CTCFL KO mice display smaller testes and increased sperm cell death, respectively [START_REF] Mannan | Male Mice Lacking the Theg (Testicular Haploid Expressed Gene) Protein Undergo Normal Spermatogenesis and Are Fertile1[END_REF][START_REF] Suzuki | Expression of a Testis-Specific Form of Gal3st1 (CST), a Gene Essential for Spermatogenesis, Is Regulated by the CTCF Paralogous Gene BORIS[END_REF].

At a mechanistic level, only the functions of a few CTAs have been described during spermatogenesis.

Some are involved in cell cycle progression, such as NY-ESO-1, which acts together with members of the MAGE family [START_REF] Thomas | NY-ESO-1 Based Immunotherapy of Cancer: Current Perspectives[END_REF]. MAGEA1 has been further identified as a transcriptional repressor, recruiting histone deacetylase during the early stages of spermatogenesis [START_REF] Laduron | MAGE-A1 interacts with adaptor SKIP and the deacetylase HDAC1 to repress transcription[END_REF]. Similar activity was associated with SSX in both gametogenesis [START_REF] Babatunde | Cancer/Testis genes in relation to sperm biology and function[END_REF] and tumorigenesis, and it is regulated by a differential cellular localization [START_REF] Wang | Subnuclear distribution of SSX regulates its function[END_REF]. In contrast, the non-X CTAs that are expressed in the late stages of spermatocyte differentiation play a role in meiosis. For example, SCP1 generates the transversal filaments of the synaptonemal complex [START_REF] Meuwissen | Human Synaptonemal Complex Protein 1 (SCP1): Isolation and Characterization of the cDNA and Chromosomal Localization of the Gene[END_REF], while SPO11 induces double-strand DNA breaks to initiate the recombination events during meiosis [START_REF] Vrielynck | A DNA topoisomerase VI-like complex initiates meiotic recombination[END_REF]. Finally, ADAM2 is expressed on the spermatozoa surface to allow sperm-egg membrane binding [START_REF] Kim | Mouse Sperm Lacking ADAM1b/ADAM2 Fertilin Can Fuse with the Egg Plasma Membrane and Effect Fertilization[END_REF].

IV. SHARED FEATURES BETWEEN SPERMATOGENESIS AND CANCER: ENERGY METABOLISM SUSTAINS CELL PROLIFERATION

The very unique expression pattern of CTAs suggests their involvement in common mechanisms between spermatogenesis and tumorigenesis. Beyond the processes involved in cell proliferation, immortalization, invasion, and migration (see reviews by [START_REF] Simpson | Cancer/testis antigens, gametogenesis and cancer[END_REF][START_REF] Whitehurst | Cause and Consequence of Cancer/Testis Antigen Activation in Cancer[END_REF])),

we wish to raise here the fact that spermatogenesis and tumorigenesis similarly involve a major metabolic shift, likely involving major mitochondrial regulations. Indeed, during carcinogenesis, cancer cells undergo a metabolic switch from aerobic respiration towards aerobic glycolysis, the socalled Warburg effect. This leads to a massive production of pyruvate from the available glucose.

Some glycolysis intermediates are then used for the anabolism dedicated to cell proliferation.

Moreover, pyruvate is converted to lactate, which is excreted in the tumor environment and used by surrounding cells to produce large amounts of pyruvate, and subsequently ATP through the oxidative phosphorylation (OXPHOS) process in mitochondria [START_REF] Feron | Pyruvate into lactate and back: From the Warburg effect to symbiotic energy fuel exchange in cancer cells[END_REF]. In most cancer cells, this process is further related to cell death resistance, insensitivity to growth suppressors, invasion, migration and angiogenesis [START_REF] Hanahan | Hallmarks of Cancer: The Next Generation[END_REF]. Importantly, in tumors (Figure 1-left panel), both proliferating glycolytic cells and their surrounding oxidative partners are working synergistically to efficiently promote tumor development.

Interestingly, during spermatogenesis, a similar situation is encountered in the seminiferous tubules, with lactate gradient extending from the Sertoli cells to the center of the tubules. In the periphery, Sertoli cells, which contribute to the Blood-Testis Barrier (BTB), have access to blood glucose to generate large amounts of lactate via the glycolysis. Similarly, the spermatogonia and primary spermatocytes located at the basal compartment generate lactate by glycolysis [START_REF] Ramalho-Santos | Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells[END_REF], needed for the next steps of spermatogenesis. Indeed, deeper in the tubule, spermatocytes, spermatids, and spermatozoa, which compose the adluminal compartment, use lactate as an energy substrate to produce ATP by OXPHOS (Figure 1-right panel), forcing mitochondria to engage in this process. Thus, the tubule fluid is enriched in lactate and poor in glucose, promoting a high oxidative activity in spermatozoa, despite having reduced access to oxygen [START_REF] Boussouar | Lactate and energy metabolism in male germ cells[END_REF][START_REF] Rato | Metabolic regulation is important for spermatogenesis[END_REF]. In conclusion, both spermatogenesis and tumorigenesis orchestrate a metabolic synergy between two cell populations: one producing lactate from glucose, another one using the available lactate to produce ATP by OXPHOS.

V. PREDICTION OF CTAS ACTING ON MITOCHONDRIA

To identify and predict if some CTAs interfere with mitochondrial physiology, we used two in silico approaches. The first one identified all the CTAs that are already described in bibliographic databases as impacting on mitochondrial functions. The second, predicted all CTA protein sequences that included a mitochondria targeting sequence (MTS), underlying their potential mitochondrial localization.

V.1. FIRST APPROACH: IDENTIFICATION OF CTAS ACTING ON MITOCHONDRIA

To identify CTAs that can potentially act on mitochondria, we designed a four steps selective process based on online databases (Figure 2). In Step 1, using the CTA-Database (http://www.cta.lncc.br/) [START_REF] Almeida | CTdatabase: a knowledge-base of high-throughput and curated data on cancer-testis antigens[END_REF] we listed the 276 genes that are encoding CTAs. In

Step 2, we analyzed the sequence of all refseq genes and transcripts to eliminate pseudogenes and lncRNA (Supplementary Table 1, highlighted in gray), reducing the list to 243 CTA genes. In Step 3, using the integrated web resource Mitominer [START_REF] Smith | MitoMiner v3.1, an update on the mitochondrial proteomics database[END_REF], which lists all proteins with mitochondrial functions (Supplementary Table 2), we reduced the CTA list to the proteins that were reported to act on mitochondria, by at least one of the four following databases:

-Gene Ontology (GO; http://geneontology.org), listing proteins associated with mitochondrial processes [START_REF] Ashburner | Gene Ontology: tool for the unification of biology[END_REF]; "The Gene Ontology Resource," 2019), -MitoCarta, (https://www.broadinstitute.org/files/shared/metabolism/mitocarta/human.mitocarta2.0.html), a database including 1157 genes encoding mammalian mitochondrial proteins [START_REF] Calvo | MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins[END_REF], -Integrated Mitochondrial Protein Index (IMPI, http://www.mrc-mbu.cam.ac.uk/impi) providing a collection of 1626 human genes encoding proteins with known (1184) or predicted (442) mitochondrial localization [START_REF] Smith | MitoMiner v3.1, an update on the mitochondrial proteomics database[END_REF], -Human Protein Atlas (http://www.proteinatlas.org), listing proteins located in mitochondria using immunofluorescence imaging [START_REF] Uhlen | Tissue-based map of the human proteome[END_REF]. This led to the identification of 10 CTAs: FTHL17, KIAA0100, POTEH, DDX43, MAGEA11, GPAT2, FATE1, COX6B2, SPATA19 and PAGE1 (Table 1, figure 2). With the last Step 4, by Pubmed abstract mining, we further reduced this list to five CTAs that were confirmed as acting on mitochondria.

Table1: The 10 identified mitoCTAs in step 3 described in Figure 2. [START_REF] Bera | Five POTE paralogs and their splice variants are expressed in human prostate and encode proteins of different lengths[END_REF][START_REF] Liu | POTEH hypomethylation, a new epigenetic biomarker for glioma prognosis[END_REF] V.2. SECOND APPROACH: IDENTIFICATION OF CTA ISOFORMS WITH A PREDICTED MITOCHONDRIAL TARGETING SEQUENCE.

Gene

Among the estimated 2000 different proteins that compose the mitochondrial proteome, 13 are encoded and synthetized within the mitochondria, while the remaining are encoded by the nuclear genome, synthesized in the cytoplasm and then imported into mitochondria. The vast majority of these imported proteins contains the MTS, an N-terminal sequence with particular structure and hydrophobicity [START_REF] Roise | Recognition and binding of mitochondrial presequences during the import of proteins into mitochondria[END_REF] which can be predicted in silico. We used four online prediction softwares: MitoFates [START_REF] Fukasawa | MitoFates: Improved Prediction of Mitochondrial Targeting Sequences and Their Cleavage Sites[END_REF], MitoProt-II [START_REF] Claros | Computational Method to Predict Mitochondrially Imported Proteins and their Targeting Sequences[END_REF], iPSORT [START_REF] Bannai | Extensive feature detection of N-terminal protein sorting signals[END_REF] and TargetP [START_REF] Almagro Armenteros | Detecting sequence signals in targeting peptides using deep learning[END_REF]. Two of these tools, MitoFates and iPSORT, give clearcut results as "YES" if a MTS is predicted, and "NO" if it is absent. The two others, MitoProt-II and TargetP, provide a probability score, for which we selected the thresholds of 0.4 and 0.3 respectively.

To identify CTAs encoding for isoforms with a MTS, thus potentially located into mitochondria (mitoCTAs), we first listed all the mRNA transcripts encoded by the 243 CTAs analyzed in the previous screening (Figure 3A). This led to the identification of 655 transcripts. Some CTAs encode a single mRNA variant, while others encode multiple mRNA variants, resulting from alternate splicing combinations. For example, the PTPN20 gene encodes as many as 55 different mRNAs variants and protein isoforms. We then ran the four prediction programs for each of the 655 corresponding protein sequences (Supplementary table 3). We identified 143 isoforms encoded by 67 CTAs, which are predicted to harbor a MTS by at least one of the four programs (Supplementary table 4). Figure 3B represents a Venn diagram summarizing all results and their intersections. Notably, the MitoProt-II and iPSORT softwares led to larger numbers of isoforms with a predicted MTS, and an intersection including 58 different isoforms, encoded by 33 CTA genes. Interestingly, some CTAs, such as CEP55, GAGE12G, RNF17, SPEF2 and TSAG10, encode different isoforms that are all predicted to harbor a MTS. However, other such as, TSPY1, CTNNA2, CTCFL and ODF2, encode multiple isoforms among which only a few are potentially addressed to mitochondria. The last case illustrates the predominant role of mRNA alternate splicing in targeting proteins to different cell compartments.

VI. CTAS FUNCTIONS INTO MITOCHONDRIA

With the first approach based on bibliographical data mining, we identified 5 CTAs acting on mitochondria: SPATA19, GPAT2, FATE1, COX6B2 and KIAA0100.

SPATA19 (Spermatogenesis Associated 19

) is a small mitochondrial protein of 154 amino acids (aa), with a 20 aa MTS. During spermatogenesis, SPATA19 is expressed in round spermatids and in the midpiece of the sperm tail. Study of a germ cell-specific Spata19 KO mouse revealed that this protein is required for appropriate flagellum formation and sperm motility, while the sperm head and the nuclear DNA integrity is maintained. These defects are related to mitochondrial cristae disorganization and complex IV assembly reduction, leading to decreased ATP production [START_REF] Mi | Spata19 is critical for sperm mitochondrial function and male fertility[END_REF]. In prostate cancer [START_REF] Wong | Cancer/testis antigen SPATA19 is frequently expressed in benign prostatic hyperplasia and prostate cancer[END_REF] and basal cell carcinoma (S. Ghafouri-Fard et al., 2010), SPATA19 expression is correlated with poor prognosis.

GPAT2 is one of the four isoforms of the glycerol-3-phosphate acyltransferases (GPAT1-4) required for de novo synthesis of glycolipids [START_REF] Garcia-Fabiani | Glycerol-3-phosphate acyltransferase 2 is essential for normal spermatogenesis[END_REF]. These enzymes show different tissue expression patterns, subcellular localization, and substrate preferences. Among them, GPAT2

expression is restricted physiologically to testis, and is overexpressed in various cancer including melanoma, prostate and breast cancers [START_REF] Pellon-Maison | Glycerol-3-Phosphate Acyltranferase-2 Behaves as a Cancer Testis Gene and Promotes Growth and Tumorigenicity of the Breast Cancer MDA-MB-231 Cell Line[END_REF]. GPAT2 is located in the outer mitochondrial membrane, interacts with small non-coding RNAs and it is essential for the primary Piwi-interacting RNAs (piRNAs) biogenesis. This promote cell proliferation and resistance to staurosporin induced apoptosis [START_REF] Shiromoto | GPAT2, a mitochondrial outer membrane protein, in piRNA biogenesis in germline stem cells[END_REF].

FATE1 (Fetal and Adult Testis Expressed 1) is a 183 aa protein that inhibits a pro-apoptotic pathway, by preventing BIK (BCL2-Interacting Killer) accumulation [START_REF] Maxfield | Comprehensive functional characterization of cancer-testis antigens defines obligate participation in multiple hallmarks of cancer[END_REF], thus promoting cell survival. FATE1 is localized at the contact sites between mitochondria and the endoplasmic reticulum (ER), the so-called mitochondrial associated membranes (MAM), due to the presence of a domain at its C-terminus required for its targeting to the mitochondrial compartment. FATE1 disrupts the ER/mitochondrial contacts, in order to prevent Ca 2+ uptake by mitochondria, thus leading to an inhibition of Ca 2+ -dependent apoptosis [START_REF] Doghman-Bouguerra | FATE1 antagonizes calcium-and drug-induced apoptosis by uncoupling ER and mitochondria[END_REF]. The expression of FATE1 is modulated by EWSR1-FLI1, which is essential for Ewing sarcoma survival [START_REF] Gallegos | EWSR1-FLI1 Activation of the Cancer/Testis Antigen FATE1 Promotes Ewing Sarcoma Survival[END_REF].

In addition to its function in apoptosis, the presence of circulating antibodies directed against FATE1 in patients with adrenocortical carcinomas was reported. FATE1 mRNA expression levels in these patients are associated with high steroidogenic gene expression, immune cell depletion and worse prognosis [START_REF] Doghman-Bouguerra | Cancer-testis Antigen FATE1 Expression in Adrenocortical Tumors Is Associated with A Pervasive Autoimmune Response and Is A Marker of Malignancy in Adult, but Not Children[END_REF]. More recently, it was reported that FATE1 was differentially expressed between long-lived versus short-lived bats [START_REF] Huang | Genetic variation between long-lived versus short-lived bats illuminates the molecular signatures of longevity[END_REF], suggesting a putative role in aging, which, however, needs to be further investigated.

COX6B2 (Cytochrome c Oxidase VIb-2), is a testis specific subunit of the cytochrome c oxidase (COX) which is the complex IV of the mitochondrial electron transport chain. COX6B2 is expressed in all steps of spermatogenesis to support the specific energy needs in testis [START_REF] Hüttemann | Cytochrome c oxidase of mammals contains a testes-specific isoform of subunit VIb-the counterpart to testes-specific cytochrome c?[END_REF] and identified as an OXPHOS-related gene signature of lung adenocarcinoma [START_REF] Xu | Development and validation of an oxidative phosphorylation-related gene signature in lung adenocarcinoma[END_REF]. It was reported that COX6B2 facilitates the assembly of complex IV. The inhibition of COX6B2 in three pancreatic ductal adenocarcinoma cell lines affects specifically complex IV activity without increasing glycolysis or subsequently altering cell growth [START_REF] Nie | COX6B2 drives metabolic reprogramming toward oxidative phosphorylation to promote metastasis in pancreatic ductal cancer cells[END_REF]. In this cancer model, COX6B2

maintains mitochondrial respiration to promote metastatic capacity rather than to increase growth capacity. Similarly, in lung adenocarcinoma cells, COX6B2 enhances OXPHOS activity and cell proliferation even in low O2 environment. However, in this cell model, the loss of COX6B2 surprisingly induced cell apoptosis indicative of a strong dependency on OXPHOS activity [START_REF] Cheng | Sperm-specific COX6B2 enhances oxidative phosphorylation, proliferation, and survival in human lung adenocarcinoma[END_REF]. Thus, it appears that the biological role of COX6B2 might be cell-type specific, as its inhibition in other cell types induces cell death as well [START_REF] Maxfield | Comprehensive functional characterization of cancer-testis antigens defines obligate participation in multiple hallmarks of cancer[END_REF].

KIAA0100 also known as BCOX1 (Breast Cancer Over-eXpressed 1), was initially characterized in breast carcinomas [START_REF] Song | Identification of BCOX1, a novel gene overexpressed in breast cancer[END_REF] with an expression negatively regulated by the microRNA miR-195. KIAA0100 is involved in cell proliferation, migration and invasion [START_REF] Guo | MicroRNA-195 suppresses tumor cell proliferation and metastasis by directly targeting BCOX1 in prostate carcinoma[END_REF], but its specific function remains unknown. In silico sequence analyses of KIAA0100 isoforms revealed a signal peptide, a transmembrane domain and four domains related to FMP27 (Found in mitochondrial proteome protein 27) [START_REF] Sickmann | The proteome of Saccharomyces cerevisiae mitochondria[END_REF], suggesting its localization in mitochondria [START_REF] Cui | Bioinformatic prediction and functional characterization of human KIAA0100 gene[END_REF].

Thus, summing up the information collected on these 5 CTAs interacting with mitochondrial physiology, it appears that the regulation of i) the mitochondrial membrane structures and their interaction with MAMs, ii) the respiratory complex IV activity, iii) the biogenesis of non-coding RNAs and iv) the apoptosis, are key mitochondrial processes related to spermatogenesis and cancer.

In the second approach, aiming at the identification of CTAs exported to mitochondria, we found 67 CTA genes, generating 143 mRNA variants encoding protein isoforms with a potential MTS. Among them, only two (SPATA19 and KIAA0100) were also identified in the first approach. Firstly, MitoprotII predicted the presence of a MTS in the two transcripts encoded by SPATA19. This prediction is in adequacy with previous reports using rats (Doiguchi et al., 2002b), mouse and human models (Doiguchi et al., 2002a;[START_REF] Matsuoka | Cloning and characterization of a mouse spergen-1 localized in sperm mitochondria[END_REF] showing the presence of a N-terminus sequence corresponding to a MTS. Secondly, 5 out of the 7 transcripts encoded by KIAA0100 were predicted to have a MTS using MitoprotII, and 1 out of the 7 transcripts was predicted to have a MTS using iPSORT. Again, this finding is consistent with a previous bioinformatics study, showing that KIAA0100 harbors 4 domains and one of them, located at N-terminus, is a MTS. This domain is already identified in the yeast mitochondrial protein FMP27 [START_REF] Zhong | KIAA0100 Modulates Cancer Cell Aggression Behavior of MDA-MB-231 through Microtubule and Heat Shock Proteins[END_REF].

However, our second approach did not identify the three other mitochondrial CTAs described in the first approach, COX6B2, FATE1 and GPAT2, because none of them harbor a MTS. This lack of concordance between the two applied approaches can be explained based on their sequence. Firstly, COX6B2, is one subunit of the complex IV that is imported into the mitochondria through the Mia40/CHCHD4 machinery. Thus, COX6B2 displays a (CX9C)2 motif required for its import into mitochondria [START_REF] Modjtahedi | Mitochondrial Proteins Containing Coiled-Coil-Helix-Coiled-Coil-Helix (CHCH) Domains in Health and Disease[END_REF]. Secondly, FATE1 is localized at the ER-mitochondria contacts. It was reported that FATE1 C-terminal domain (aa 125-183) is sufficient for its targeting to mitochondria. Three basic residues within this C-terminal domain are essential for this localization [START_REF] Doghman-Bouguerra | FATE1 antagonizes calcium-and drug-induced apoptosis by uncoupling ER and mitochondria[END_REF]. Finally, GPAT2 is localized at the mitochondrial outer membrane (MOM) and reported to have two transmembrane regions (sequences Ala306-Tyr332 and Val450-Leu472) required for its insertion in MOM and both N-and C-terminus are facing the cytosolic compartment [START_REF] Nakagawa | Membrane topology of murine glycerol-3-phosphate acyltransferase 2[END_REF].

Beside these, among the identified 67 CTAs genes predicted to encode the 143 isoforms with a potential MTS, 33 have completely unknown functions, including 16 genes of the GAGE family.

Conversely, 34 other CTAs have been partially characterized at the function level. Importantly, except KIAA0100 and SPATA19, none of these 67 mitoCTAs has yet been ascribed to a mitochondrial localization or function. Their involvement in mitochondrial physiology can only be inferred from the analysis of their primary protein sequence or from the characterized isoforms generated by the same genes but located in a different cell compartment. Indeed, in eukaryotic cells, one protein can be located in several organelles, a process termed dual targeting or dual localization. This phenomenon increases the number of cellular functions without increasing the number of genes. Several distinct mechanisms achieve this dual localization, such as mRNA alternate splicing leading to a specific transcript encoding a MTS, and alternative transcription initiation. In addition, a single protein sequence could mediate dual targeting into different compartments, a process termed ambiguous targeting signal. Indeed, targeting sequences meant for protein export in mitochondria, ER and chloroplasts, share common features, including the N-terminal position, the α-helical structure and the hydrophobicity (reviewed in [START_REF] Yogev | Dual targeting of mitochondrial proteins: Mechanism, regulation and function[END_REF]). Finally, some proteins can harbor two distinct targeting signals, one at the N-terminus and another at the C-terminus. For example, in the 30 Nterminal amino acids of LpSOD, two targeting domains can be distinguished: the signal peptide (SP)

and the transit peptide (TP). Also, LpSOD contains a potential type-1 peroxisome-targeting signal (PTS1) located at its C-terminus [START_REF] Bodył | Analysis of the targeting sequences of an iron-containing superoxide dismutase (SOD) of the dinoflagellate Lingulodinium polyedrum suggests function in multiple cellular compartments[END_REF].

By reviewing the available data concerning the 34 mitoCTAs with known structure or function, we identified five clusters of processes. A first cluster of mitoCTAs consists of proteins acting on nuclear DNA maintenance. They include the PRM1 and PRM2 protamines that substitute for histones to stabilize the DNA, as well as HORMAD1 and HORMAD2 that release DNA constraints through double strand breaks. In addition, we identified, a set of proteins involved in the structure and organization of chromosomes such as, FMR1NB, SYCP1, SYCE1, BRDT, CTCFL and OIP5.

Interestingly, the latter 4 mitoCTAs have only some alternative isoforms that are predicted to be imported into mitochondria. Whether these proteins also act on mitochondrial DNA structure and maintenance remains to be experimentally elucidated.

A second cluster includes 5 mitoCTAs (DDX43, MAEL, PIWIL1, PIWIL2 and RNF17) that are related to the RNA metabolism and in particularly Piwi-RNAs (piRNAs), for which a substantial mitochondria contribution in their processing has already been disclosed [START_REF] Aravin | piRNAs Meet Mitochondria[END_REF].

Importantly, for MAEL and PIWIL2, specific isoforms are present in both mitochondria and other cell compartments, as only one out of their three isoforms harbors an MTS. Thus, whether specific regulations of piRNAs by mitochondria are common between spermatogenesis and tumorigenesis remains to be elucidated.

A third cluster is related to genes possibly involved in the mitochondrial transport within the cell, based on their potential interaction with kinesin and dynein motors [START_REF] Pilling | Kinesin-1 and Dynein Are the Primary Motors for Fast Transport of Mitochondria in Drosophila Motor Axons[END_REF]. Three of these mitoCTAs are microtubule-dependent molecular motors that can be localized into mitochondria: SPEF2, a linker protein for dynein1 [START_REF] Lehti | SPEF2 functions in microtubule-mediated transport in elongating spermatids to ensure proper male germ cell differentiation[END_REF], two isoforms of KIF2C, a kinesin-like protein and one isoform of SPAG4, which interacts with NESPRIN3 [START_REF] Ji | Sperm-associated antigen 4 (SPAG4) as a new cancer marker interacts with Nesprin3 to regulate cell migration in lung carcinoma[END_REF]. In addition, some isoforms of ODF2 might also be restricted to mitochondria, although its function has been related to the biogenesis of centrosomes. These observations question the potential role of this group of mitoCTAs in mitochondrial dynamics and localization within the cell.

A fourth cluster involves CTAs potentially regulating cell signaling, and in particular the calcium fluxes at the contact sites between ER and mitochondria, MAMs, which are involved in Ca 2+ signaling, lipid synthesis and mitochondrial biogenesis regulation [START_REF] Rowland | Endoplasmic reticulum-mitochondria contacts: function of the junction[END_REF]. Here, we identified 3 mitoCTAs: CALR3, a calcium-binding chaperone protein, CABYR, a calcium-binding tyrosine phosphorylation-regulated protein, and LIP1 a phospholipase that could be directed to the mitochondrial side of the MAMs to regulate calcium fluxes. The two first mitoCTAs are indispensable for the development of sperm fertilizing ability [START_REF] Ikawa | Calsperin Is a Testis-specific Chaperone Required for Sperm Fertility[END_REF][START_REF] Young | CABYR is essential for fibrous sheath integrity and progressive motility in mouse spermatozoa[END_REF], but only CABYR has been involved in tumorigenesis. Therefore, it will be interesting to track the localization of CALR3, CABYR and LIP1 in living cells and to assess their role in calcium signaling. Again, additional different CABYR isoforms could be located alternatively outside mitochondria, as only 3 out of its 7 isoforms are predicted to include a MTS.

Additional mitoCTAs, such as DCAF12, PRAME and TSPY1 could play a role in particular signaling processes in the mitochondria. For example, TSPY1 has been shown to regulate p53 levels, a protein that is also present in mitochondria.

A fifth cluster involves the TSAG10, SPAC13 and CTNNA2 genes, which play a role in sperm cell adhesion. However, their functions in mitochondria remain to be elucidated.

VII. DISCUSSION AND OPEN QUESTIONS

In summary, with our two approaches, we identified 70 mitoCTAs among the 276 known CTAs: 67

CTAs predicted to harbor a MTS and 3 without a MTS. Their expression might be related to specific mitochondrial functions involved in spermatogenesis in physiological condition, and in tumorigenesis in a pathological condition. Initially, our hypothesis concerning the possible role of CTAs in mitochondria was based on the fact that both spermatogenesis and tumorigenesis involved a common major metabolic shift, requiring an adaptation of the respiratory chain to available substrates. Indeed, our findings suggest that complex IV of the respiratory chain modifies its subunits composition to adapt its efficacy and drive the production of ATP through the OXPHOS process, in order to consume the high amounts of lactate available in the microenvironment. However, this potential metabolic adaptation only involves two of our identified mitoCTAs: SPATA19 and COX6B2. Therefore, the many other mitoCTAs described here might foster equally important functions for spermatogenesis and tumorigenesis, such as the organization of the mitochondrial genome, the control of non-coding RNAs, as the piRNAs, and signaling pathways, mostly related to calcium fluxes between the ER and the mitochondria.

Thus, altogether, these processes could regulate the expression of the mitochondrial genome, the activity of mitochondrial matrix enzymes involved in the lactate catabolism and eventually regulate the whole cell metabolism required during cell proliferation, immortalization, invasion and migration.

If some of these processes have already been associated to spermatogenesis, such as the piRNA turnover, their involvement in the metabolic adaptations in tumorigenesis and spermatogenesis remains to be elucidated. Ultimately, the contribution of many mitoCTAs to modulations in mitochondrial membranes' shape and composition, as well as to the cellular distribution of mitochondria during spermatogenesis and cancer development should be further investigated.

To conclude, all these predicted data generate a tremendous number of questions on mitoCTAs, and the following three challenges should help in understanding their precise roles in the cell:

The first one consists in determining the precise cellular localization of mitoCTAs. Indeed, among them, only 5 have been proven to act on mitochondrial functions, while other are predicted to harbor a MTS by at least one of the four prediction tools we used. This implies that the first obvious task to address in the future consists in determining the precise localization of all mitoCTAs identified here.

Thus, we could refine the listing of the true mitoCTAs and define in which mitochondrial subcompartment they exert their functions.

The second challenge is to unravel the function of each mitoCTAs in mitochondrial physiology and their interaction with the microenvironment. This is a key process in understanding why mitoCTAs are crucial during spermatogenesis and cancer development. Moreover, it should shed light on unexpected mitochondrial particularities that are restricted to germ and cancer cells and absent in somatic cells.

The last challenge is to understand how the expression of mitoCTAs is controlled, by defining the timing of expression for each mitoCTA during the different stages of spermatogenesis and tumorigenesis, and by understanding how the biosynthesis and stability of their mRNAs is regulated at the molecular level.

Together, these data should bring insights on novel mitochondrial functions that are specific to germ and tumor cells. Importantly, this should provide new targets for the design of specific cancer treatments that will not affect the integrity of somatic cells. Step 2) Using official gene symbol and Refseq we excluded 5 pseudogenes, 8 ncRNA and 20 genes with wrong official CTA gene symbol, leading to a list of 243 CTAs.

Figures' legends

Step 3) Among the 243 CTAs, we identified 10 CTAs linked to mitochondrial functions in at least one of the following databases: MitoCarta, GO Annotation, IMPI score (threshold score >0,5) and the Human Protein Atlas.

Step 4) Using PubMed abstract mining, we identified 5 CTAs; COX6B2, GPAT2, SPATA19, FATE1 and KIAA0100 involved in mitochondrial functions. (B) Venn diagram distribution of the 10 CTAs identified in step 3, using the MitoCarta, GO Annotation, IMPI score and Human Protein Atlas databases.

Figure 1 :

 1 Figure 1: Schematic illustration of a metabolic synergy between two cell populations during tumorigenesis and spermatogenesis.During tumorigenesis and spermatogenesis, lactate production, shuttling and gradient are defining energetic limiting parameters, enforcing a symbiotic cooperation between different cell populations. Left panel: during tumorigenesis, under hypoxia condition in tumors, cancer cells located at the center use glycolysis as the main source of energy and release lactate in the microenvironment to support energy production by normoxic tumor cells, thus contributing to their proliferation and tumorigenesis. Right panel: during spermatogenesis, Sertoli cells, which use glycolysis, support spermatogenesis by supplying lactate to cells located after the blood-testis barrier. Lactate is taken up by spermatocytes I and spermatocytes II and used as the source for ATP production by mitochondria. The cells shown in this figure were created using images modified from Servier Medical Art Commons Attribution 3.0 Unported License. (http://smart.servier.com).

Figure 2 :

 2 Figure 2: Identification of CTAs acting on mitochondria (A) Description of the four steps workflow to identify the CTAs reported to act on mitochondria: Step 1) Using the CTDatabase, we listed 276 known CTAs.Step 2) Using official gene symbol and Refseq we excluded 5 pseudogenes, 8 ncRNA and 20 genes with wrong official CTA gene symbol, leading to a list of 243 CTAs.Step 3) Among the 243 CTAs, we identified 10 CTAs linked to mitochondrial functions in at least one of the following databases: MitoCarta, GO Annotation, IMPI score (threshold score >0,5) and the Human Protein Atlas. Step 4) Using PubMed abstract mining, we identified 5 CTAs; COX6B2, GPAT2, SPATA19, FATE1 and KIAA0100 involved in mitochondrial functions. (B) Venn diagram distribution of the 10 CTAs identified in step 3, using the MitoCarta, GO Annotation, IMPI score and Human Protein Atlas databases.

Figure 3 :

 3 Figure 3: Identification of mitoCTA isoforms with a predicted MTS (A) Description of the three steps workflow to identify the CTAs with a predicted MTS. Step 1) Using the CTDatabase, we listed 276 CTAs.Step 2) we identified 655 isoforms encoded by the mRNAs of the 243 CTAs (see Figure1).Step 3) Using iPSORT, TargetP mTP, MitoFates and MitoProt-II online tools, we identified among them143 isoforms encoded by 67 CTAs, for which a MTS predicted was predicted by at least one of the 4 tools. (B) Venn diagram representing the distribution of the 143 isoforms encoded by the 67 CTAs, with a MTS predicted by 1 or more of the 4 prediction tools: iPSORT (Green), Mitoprot II (Red), MitoFates (Brown) and TargetP mTP (Purple). Each CTA gene symbol name is followed by the number of isoforms with a MTS, among the total number of isoforms encoded by the CTA. Example: SPACA3 1/3 means that SPACA3 encodes for 3 isoforms, with only one predicted with a MTS. Identified CTAs in the first approach are highlighted on yellow.

Acknowledgements

We acknowledge Dr. Layon for promoting scientific discussions and Sheena M Aris, Shar-Yin N Huang and Brunilde M. Gril for the critical reading of the manuscript.

Funding

This work was supported by the Université d'Angers, CHU d'Angers, the Région Pays de la Loire and Angers Loire Métropole.

Contribution of each author to the study

1) Conception, design and writing of the review: Jade Aurrière, Guy Lenaers and Salim Khiati.

2) Acquisition and analysis of the molecular and bibliographic data: Jade Aurrière, David Goudenège, Magalie Boguenet.

3) Drafting a significant portion of the manuscript and figures: Jade Aurrière, Olivier R. Baris, Pascale May-Panloup, Guy Lenaers and Salim Khiati.

Conflicts of Interest

All authors declare that they have no conflict of interest with the publication of the review presented here.