Piia Liigand
email: piia.liigand@ut.ee

Agnes Heering

Karl Kaupmees

Ivo Leito

Marion Girod

Rodolphe Antoine

Anneli Kruve

The evolution of electrospray generated droplets is not affected by ionization mode

Ionization efficiency and mechanism in ESI is strongly affected by the properties of mobile phase.

The use of mobile phase properties to accurately describe droplets in ESI source is convenient but may be inadequate as the composition of the droplets is changing in the plume due to electrochemical reactions occurring in the needle tip as well as continuous drying and fission of droplets. Presently, there is paucity of research on the effect of the polarity of the ESI mode on mobile phase composition in the droplets. In this paper the change in the organic solvent content, pH and droplet size are studied in the ESI plume in both ESI+ and ESI-ionization mode. We introduce a rigorous waythe absolute pH (pH abs H 2 O )to describe pH change in the plume that takes into account organic solvent content in the mobile phase. pH abs H 2 O enables comparing acidities of ESI droplets with different organic solvent contents. The results are surprisingly similar for both ionization modes indicating that the dynamics of the change of mobile phase properties is independent from the ESI mode used. This allows us to conclude, that the evolution of ESI droplets first of all proceeds via the evaporation of the organic modifier and to a lesser extent via fission of smaller droplets from parent droplets. Secondly, our study shows that qualitative findings related to the ESI process obtained on the ESI+ mode can almost directly be applied also in the ESI-mode.

Introduction

It has been observed that different solvents have different influence on ionization in electrospray ionization mass spectrometry (ESI/MS) analysis [START_REF] Cech | Practical implications of some recent studies in electrospray ionization fundamentals[END_REF][START_REF] Cole | Electrospray and MALDI Mass Spectrometry: fundamentals, instrumentation, practicalities, and biological applications[END_REF]. Most often acetonitrile or methanol in a mixture with water are used as mobile phase in ESI. It has been shown that in many cases ESI response is higher in solutions with higher organic modifier percentage [START_REF] Kruve | Influence of mobile phase, source parameters and source type on electrospray ionization efficiency in negative ion mode: Influence of mobile phase in ESI/MS[END_REF]. This may be due to more efficient desolvation of the droplets, allowing them to reach Rayleigh limit faster and eventually generate smaller droplets more rapidly [START_REF] Zhou | Effects of solvent composition of molecular ion response in electrospray mass spectrometry: investigation of the ionization process[END_REF]. pH of the mobile phase also influences ionization in the ESI plume [START_REF] Liigand | pH Effects on Electrospray Ionization Efficiency[END_REF]. It has been established that in general basic analytes provide higher sensitivity with more acidic mobile phase in ESI positive mode (ESI+) and acidic analytes with more basic mobile phase in ESI negative mode (ESI-) [START_REF] Cole | Electrospray and MALDI Mass Spectrometry: fundamentals, instrumentation, practicalities, and biological applications[END_REF].

It has also been shown that protonated forms of basic analytes can frequently be observed even if the pKa (of the protonated analyte) is far below the solutions pH [START_REF] Zhou | Protonation in electrospray mass spectrometry: wrong-way-round or right-way-round?[END_REF][START_REF] Oss | Electrospray Ionization Efficiency Scale of Organic Compounds[END_REF][START_REF] Liigand | Effect of Mobile Phase on Electrospray Ionization Efficiency[END_REF]. Similarly deprotonated forms of analytes can be observed when ESI/MS analysis is performed with acidic solutions with pH lower than the analyte pKa [START_REF] Zhou | Protonation in electrospray mass spectrometry: wrong-way-round or right-way-round?[END_REF][START_REF] Kruve | Negative Electrospray Ionization via Deprotonation: Predicting the Ionization Efficiency[END_REF]. Therefore pH is an important parameter influencing ionization.

In terms of the mobile phase, the initial composition is generally used to describe the ESI process as the actual composition in the plume is difficult to measure. However, it has been shown that solvent pH [START_REF] Zhou | Investigation of the Electrospray Plume by Laser-Induced Fluorescence Spectroscopy[END_REF][START_REF] Zhou | Profiling pH Changes in the Electrospray Plume[END_REF][START_REF] Girod | Relation between charge state distributions of peptide anions and pH changes in the electrospray plume. A mass spectrometry and optical spectroscopy investigation[END_REF][START_REF] Girod | Basic Vapor Exposure for Tuning the Charge State Distribution of Proteins in Negative Electrospray Ionization: Elucidation of Mechanisms by Fluorescence Spectroscopy[END_REF], organic modifier content [START_REF] Liigand | Effect of Mobile Phase on Electrospray Ionization Efficiency[END_REF][START_REF] Girod | Profiling an electrospray plume by laserinduced fluorescence and Fraunhofer diffraction combined to mass spectrometry: influence of size and composition of droplets on charge-state distributions of electrosprayed proteins[END_REF][START_REF] Zhou | Probing Solvent Fractionation in Electrospray Droplets with Laser-Induced Fluorescence of a Solvatochromic Dye[END_REF][START_REF] Wang | Evolution of the solvent polarity in an electrospray plume[END_REF][START_REF] Hopkins | Evaporation of Ethanol/Water Droplets: Examining the Temporal Evolution of Droplet Size, Composition and Temperature[END_REF], and droplet size [START_REF] Girod | Profiling an electrospray plume by laserinduced fluorescence and Fraunhofer diffraction combined to mass spectrometry: influence of size and composition of droplets on charge-state distributions of electrosprayed proteins[END_REF][START_REF] Hopkins | Evaporation of Ethanol/Water Droplets: Examining the Temporal Evolution of Droplet Size, Composition and Temperature[END_REF][START_REF] Wortmann | Shrinking droplets in electrospray ionization and their influence on chemical equilibria[END_REF][START_REF] Soleilhac | Correlating Droplet Size with Temperature Changes in Electrospray Source by Optical Methods[END_REF] change along the plume. The complex dynamics of the ESI process make the transition of ions from solution-phase to gas-phase difficult to model. Atomistic molecular dynamics methods for such simulations have been used [START_REF] Oh | Stability of a Transient Protein Complex in a Charged Aqueous Droplet with Variable pH[END_REF][START_REF] Sharawy | How do non-covalent complexes dissociate in droplets? A case study of the desolvation of dsDNA from a charged aqueous nanodrop[END_REF][START_REF] Ahadi | Molecular Dynamics Simulations of Electrosprayed Water Nanodroplets: Internal Potential Gradients, Location of Excess Charge Centers, and "Hopping" Protons[END_REF][START_REF] Sharawy | Characterization of "Star" Droplet Morphologies Induced by Charged Macromolecules[END_REF][START_REF] Consta | New mechanisms of macroion-induced disintegration of charged droplets[END_REF][START_REF] Malevanets | Variation of droplet acidity during evaporation[END_REF], but are usually limited to the droplets containing up to a few thousand solvent molecules. Therefore, it is advantageous to directly perform optical spectroscopic measurements of physicochemical parameters of droplets and their changes in the electrospray plume in order to correlate the ions observed in the initial sample solution to those observed in the gas phase by the mass-spectrometer.

Fluorescence excitation studies of octaethylporphyrin in the electrospray stream pioneered in using spectroscopy to describe the processes in ESI plume [START_REF] Chillier | A Mass Spectrometry and Optical Spectroscopy Investigation of Gas-phase Ion Formation in Electrospray[END_REF]. More recently several researchers [START_REF] Zhou | Investigation of the Electrospray Plume by Laser-Induced Fluorescence Spectroscopy[END_REF][START_REF] Zhou | Profiling pH Changes in the Electrospray Plume[END_REF][START_REF] Zhou | Probing Solvent Fractionation in Electrospray Droplets with Laser-Induced Fluorescence of a Solvatochromic Dye[END_REF][START_REF] Wang | Evolution of the solvent polarity in an electrospray plume[END_REF][START_REF] Hopkins | Evaporation of Ethanol/Water Droplets: Examining the Temporal Evolution of Droplet Size, Composition and Temperature[END_REF][START_REF] Wortmann | Shrinking droplets in electrospray ionization and their influence on chemical equilibria[END_REF][START_REF] Davis | Profiling an Electrospray Plume Using Surface-Enhanced Raman Spectroscopy[END_REF] have developed laser-induced fluorescence strategies to probe properties of ESI plume, e.g. solvent fractionation, solvent polarity, pH and temperature, and correlate changes with droplet size evolution along the ESI plume. Whereas these experiments were performed by mimicking electrospray sources (i.e. without actual MS measurements), there have also been studies on the aforementioned changes in combination with mass spectrometry [START_REF] Liigand | Effect of Mobile Phase on Electrospray Ionization Efficiency[END_REF][START_REF] Girod | Relation between charge state distributions of peptide anions and pH changes in the electrospray plume. A mass spectrometry and optical spectroscopy investigation[END_REF][START_REF] Girod | Basic Vapor Exposure for Tuning the Charge State Distribution of Proteins in Negative Electrospray Ionization: Elucidation of Mechanisms by Fluorescence Spectroscopy[END_REF][START_REF] Girod | Profiling an electrospray plume by laserinduced fluorescence and Fraunhofer diffraction combined to mass spectrometry: influence of size and composition of droplets on charge-state distributions of electrosprayed proteins[END_REF][START_REF] Soleilhac | Correlating Droplet Size with Temperature Changes in Electrospray Source by Optical Methods[END_REF].

Previous studies have measured either aqueous phase pH or the pH of the mobile phase (mixture of water phase and organic solvent) using calibration in water phase. Depending on the organic modifier, it can significantly influence the pH of the solvent mixture indicating that the conclusions drawn from the aqueous phase pH might be over-simplified. We improve upon the conventional approach by for the first time profiling the pH in the spray according to the absolute pH scale [START_REF] Himmel | Anchor Points for the Unified Brønsted Acidity Scale: The rCCC Model for the Calculation of Standard Gibbs Energies of Proton Solvation in Eleven Representative Liquid Media[END_REF].

It is expressed as pH abs

H 2 O values [START_REF] Suu | Unified pH Values of Liquid Chromatography Mobile Phases[END_REF], which enables direct comparison of the pH between different solvents and water (see Experimental section). The acidity of each solvent composition was probed in the aqueous pH ( pH w w

, refers to the "conventional" pH calibrated and measured in water) range from 3.2 to 5.2 using pH-chromic fluorescent dye as the molecular probe.

ESI+ and ESI-mode have only rarely been profiled simultaneously. Cook et al [START_REF] Zhou | Investigation of the Electrospray Plume by Laser-Induced Fluorescence Spectroscopy[END_REF] has studied the pH change in water droplets and Zenobi et al [START_REF] Wortmann | Shrinking droplets in electrospray ionization and their influence on chemical equilibria[END_REF] has compared the droplet size for acetonitrile droplets for ESI+ and ESI-. However, usually mobile phase contains both water and organic solvent and the evolution of several parameters -pH, organic modifier content, droplet sizeoccurs simultaneously.

The aim of this study is to quantify the changes occurring in the ESI plume: organic modifier percentage, pH as well as droplet size change of the "parent" ESI droplets with the diameter range of a few micrometers. For quantitative comparison of pH changes, the absolute pH is used which enables comparing different solvents and solvent compositions. Even more so, we will estimate, whether ESI polarity affects the magnitude of mobile phase properties' change in the ESI plume.

This enables for the first time to compare the processes occurring in ESI source and aids in better understanding of the differences and similarities of the two modes.

Experimental

Absolute pH measurements

The conventional pH is defined by IUPAC [START_REF] Buck | Measurement of pH. Definition, standards, and procedures[END_REF] as given in Eq. ( 1):

H a log pH   (1)
where aH is the relative activity in molal scale. This means that the zero point (standard state) of pH scale is activity of 1 mol/kg of H + solv in the given solvent (or solvent mixture). Therefore, every solvent or solvent mixture has its own pH scale and none of these scales are comparable due to unknown shifts of the zero points. To add confusion to these different pH scales there is the question of calibration, which leads to another possibility to define or name pH scales as pH , where subscript shows calibration and superscript measurement medium (s is solvent and w is water). This question is discussed in detail by Rosés [START_REF] Himmel | A Unified pH Scale for All Phases[END_REF].

Recently Himmel et al. proposed a unified acidity scale that is based on the absolute standard chemical potential abs(H + ) of the solvated proton [START_REF] Himmel | Anchor Points for the Unified Brønsted Acidity Scale: The rCCC Model for the Calculation of Standard Gibbs Energies of Proton Solvation in Eleven Representative Liquid Media[END_REF] and where the zero point of the scale is the abs(H + ) of the proton in the gas phase, which is arbitrarily set to 0 kJ mol -1 . Importantly, this zero point of the scale is universal to all possible media, thereby enabling comparison the acidities of any given media on one scale. This approach is also fully universal in the sense that it does not set any limitations to the solvation sphere of the proton.

The absolute acidities pHabs can be calculated via chemical potentials as follows:

pH abs = - 𝜇 𝑎𝑏𝑠 (H + ,solv) 𝑅𝑇 ln 10 ( 2
)
where R is the molar gas constant, and T is the absolute temperature and abs(H + ,solv) is calculated as given in Eq. ( 3)

𝜇 𝑎𝑏𝑠 (H + , solv) = ∆ solv 𝐺 o (H + ) -[pH × 𝑅𝑇 ln 10] (3)
where solvG ⦵ (H + ) is Gibbs energy of solvation and pH is the conventional pH.

As was said by IUPAC already in 1985 [START_REF] Mussini | Criteria for standardization of pH measurements in organic solvents and water + organic solvent mixtures of moderate to high permittivities[END_REF] an "intersolvental" pH scale would be ultimately referenced to water due to the indisputable key role of water as a solvent. For the same reason absolute acidity is linked to water pH scale via Gibbs energy of solvation as follows: ) values.

pH

Measurements

Absolute pH values (Table 1) were obtained by using a Metrohm 713 pH meter in differential potentiometry mode with two metal-coated glass electrodes (Laboratory of Glass Electrochemistry, St. Petersburg State University) as described in [START_REF] Suu | Unified pH Values of Liquid Chromatography Mobile Phases[END_REF]. Measurements were made at (25 ± 1) °C. Consistency standard deviation of the results was 0.01 pH units and in total 52 measurements were made. Liquid junction potentials and uncertainties were calculated as in ref [START_REF] Suu | Unified pH Values of Liquid Chromatography Mobile Phases[END_REF]. Absolute pH values were measured in bulk solutions and used as reference for calibrating . b Within-lab reproducibility estimates (uRW) can be used to evaluate the internal consistency of the measured values. c These combined standard uncertainty estimates (uC) can be used to compare the acidities of the solutions in this scale with the acidities of aqueous solutions by conventional pH measurement.

3 ESI plume profiling

MeCN content was profiled in ESI+ and ESI-for solutions initially containing 80% and 50% MeCN (v/v) from fluorescent measurements of the solvatochromic dye Nile Red [START_REF] Girod | Profiling an electrospray plume by laserinduced fluorescence and Fraunhofer diffraction combined to mass spectrometry: influence of size and composition of droplets on charge-state distributions of electrosprayed proteins[END_REF]. pH was profiled for mobile phases with initial compositions of acetonitrile/aqueous solution with pH flow rate with a KDS100 syringe pump (KD Scientific, Holliston, MA, USA).

The temperature of the droplets was not profiled in this study. However previous studies performed with methanol droplets have shown either a slight increase of temperature [START_REF] Soleilhac | Correlating Droplet Size with Temperature Changes in Electrospray Source by Optical Methods[END_REF] or some decrease [START_REF] Gibson | Fluorometric Measurement and Modeling of Droplet Temperature Changes in an Electrospray Plume[END_REF] along the plume. The temperature of the plume in the referred studies was found to be between 295 and 307 K.

A continuous laser (λ = 473 nm) emitting in a single longitudinal mode was used to profile ESI plume. The output power of the laser was around 500 mW and its beam diameter is 1. After plotting the signal intensity as a function of the scattering angle, using a homemade software, comparison with the Mie theory [START_REF] Bohren | Absorption and Scattering of Light by Small Particles[END_REF] allowed us to obtain the droplet size with accuracy better than 2.5 % due to the high sensitivity of the interference pattern to the size for µm-sized droplets.

Compounds

The First of all, the chromism of Nile Red (20 µM) was calibrated in MeCN/water binary solvent mixtures. Variation in the MeCN/water ratio induces a shift of the maximum emission wavelength.

The solvent composition of an unknown solution can be determined based on the λmax using the calibration curve.

For pH measurements, stock solutions containing 0.1 mM of 5( 6 . The obtained water phase solutions were then mixed in different ratios with acetonitrile so that acetonitrile content in the calibration solutions was 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%. In order to establish calibration curves, fluorescence spectra of all the obtained 54 mixtures were measured, in solution and logarithm of the ratios of fluorescence emission intensities was calculated.

Results

The results of MeCN content and pH abs H 2 O in the ESI plume for all studied mobile phases are brought in Table 2. In all cases, the MeCN content decrease across the plume is 5.3 to 7.5% (percentage point), as acetonitrile is more readily vaporized during the evaporation process of the droplets.

These changes in MeCN content in the ESI plume have been taken into account for the determination of the pH. This means for every point in the pH calibration curve the corresponding MeCN content percentage was used. For all mobile phases the ESI+ mode has somewhat smaller change in MeCN content, however the difference is insignificant. evaporation process. Note that the reported droplet size is an average of the size of droplets within a pixel, which is an area of 500 µm x 500 µm. Moreover, the droplets constituting the ESI plume are polydispersed. The droplet size distribution for each pixel is found to be Gaussian with a standard deviation σ ranging from 1 % to 2.5 %. The droplet diameter is decreasing along the plume from 4.5 µm to 2 µm for the MeCN/water 80/20 solvent mixture. For the 50/50 mixture, the initial droplet diameter is higher (4.9 µm) and the solvent evaporation is less efficient, leading to droplet diameter of 3.6 µm at x = 13 mm from the emitter tip. However, droplet size measurements demonstrate that droplet diameter change is independent of the ESI mode.

Discussion

In this paper for the first time we simultaneously measure plume parameters for ESI+ [START_REF] Wang | Evolution of the solvent polarity in an electrospray plume[END_REF][START_REF] Davis | Profiling an Electrospray Plume Using Surface-Enhanced Raman Spectroscopy[END_REF][START_REF] Gibson | Fluorometric Measurement and Modeling of Droplet Temperature Changes in an Electrospray Plume[END_REF] and ESI- [START_REF] Girod | Profiling an electrospray plume by laserinduced fluorescence and Fraunhofer diffraction combined to mass spectrometry: influence of size and composition of droplets on charge-state distributions of electrosprayed proteins[END_REF] for acidic mobile phase. Though both modes have been used in plume profiling before usually one mode has been used at a time. Due to different conditions, instrumentation, measurement method, etc the results of such measurements thus far have not allowed direct comparison of ESI+ and ESI-.

Secondly, in this paper we present the study of the most acidic mobile phase profiled so far. In ESI+ acidic mobile phases are much more often used than neutral or basic mobile phases and are therefore of great interest. The main difficulty was to find a dye with low enough pKa value, with acceptable solubility in the mobile phase and significant change of spectral features with pH. The lowest pH that could be profiling was pH w w of 3.00.

Thirdly, it is the first time plume profiling is connected with pH abs H 2 O measurements, therefore allowing to compare acidities for plumes with different organic solvent content. This on the other hand makes results of this work also comparable with future researches, independent of the mobile phase of use there.

The obtained fluorescence spectra is an average of droplets present in the size of the laser beam (~1 mm). So the pH and mobile phase composition measured correspond to an area of ~0.78 mm² in the ESI plume and not for an unique droplet. The droplets profiled in this study are the large parent droplets (in a micrometer scale) observed in the ESI plume. They are in different stages of development: (1) approaching Rayleigh limit, (2) close to the Rayleigh limit and (3) already undergone Coulomb explosion and emitted significant amount of excess charge via formation of smaller offspring droplets. Therefore not all of the droplets are at the same stage and profiling gives us the average of these droplets.

It was observed that the mobile phase became more acidic (compared to the initial acidity) at the needle tip (x = 0 mm) in the ESI+ mode, whereas in ESI-it became more basic. The pH abs

H 2 O
measurements at this point are not yet influenced by the selective evaporation that occurs in the plume, as the measurements are made directly in the needle tip. This is also observable on Figure 1a, which shows the acetonitrile content along the plume starting from the needle tip. The change in pH abs H 2 O is caused by the electrochemical reactions taking place in the very end on the needle capillary. In ESI+ mode the electrochemical reaction creates an additional amount of protons from water or additives, while in ESI-hydroxyl ions are generated or protons are neutralized [10-12, 35, 36].

The pH abs H 2 O difference between bulk mobile phase and mobile phase on the capillary tip for ESI+ and ESI-modes in all mobile phases was up to 0.23 pH abs H 2 O units. Van Berkel et al [START_REF] Van Berkel | Changes in bulk solution pH caused by the inherent controlled-current electrolytic process of an electrospray ion source[END_REF] have previously found and calculated that the change could be in the order of 2 or more pH units. This may result from the fact, that in their study pure water without buffering agent was used.

Additionally, predictive calculations usually do not account for the shift of acid-base equilibria due to organic solvent content in the mobile phase (i.e. calculations/measurements are carried out in aqueous phase and organic phase influence is considered small) [@]. The pKa values of carboxylic acids increase markedly with higher organic modifier content therefore the generated protons are readily adsorbed by the buffering anions (that become stronger bases with the addition of organic modifier). Therefore the addition or removal of protons has a much smaller effect on the overall change on pH abs H 2 O .

The pH abs H 2 O change occurring between bulk phase and droplets in the needle tip is significant.

However the absolute value of pH abs H 2 O change is very similar for ESI+ (up to 0.23 units) and ESI-(up to 0.17 units). Therefore, the absolute differences in the changes of pH abs H 2 O occurring in the needle tip for ESI+ and ESI-are statistically insignificantly different. Based on this, it is impossible to decide over the efficiency of electrochemical reaction between ESI modes and mobile phases.

For all studied mobile phases and in both modes it was observed, that the solutions become more acidic along the plume. This is anticipated since in the spray acetonitrile is more readily vaporized and thus the content of MeCN in the droplets decreases along the plume, causing an increase of water content, resulting in increase of formic acid (the aqueous phase consists of 0.1% formic acid solution titrated with 25% ammonia solution to the desired pH) dissociation thereby turning the solution more acidic. The influence of solvent composition change on basicity of ammonia is less important because (1) basicity of bases is less influenced by adding organic solvent [START_REF] Suu | Unified pH Values of Liquid Chromatography Mobile Phases[END_REF] and [START_REF] Cole | Electrospray and MALDI Mass Spectrometry: fundamentals, instrumentation, practicalities, and biological applications[END_REF] the used pH range is far from the pKa of NH4 + . The pH change in the plume is in general similar to that expected and also previously demonstrated [START_REF] Girod | Relation between charge state distributions of peptide anions and pH changes in the electrospray plume. A mass spectrometry and optical spectroscopy investigation[END_REF]. Therefore showing that tendencies found previously for neutral and basic mobile phases are also valid for acidic mobile phases.

The decrease of pH abs MeCN content [START_REF] Girod | Profiling an electrospray plume by laserinduced fluorescence and Fraunhofer diffraction combined to mass spectrometry: influence of size and composition of droplets on charge-state distributions of electrosprayed proteins[END_REF]: both 50/50 and 80/20 mobile phases lose about 5% of acetonitrile, therefore the total acetonitrile loss (and thereby the increase in water content leading to increased dissociation of formic acid) for the latter is higher. Solvent fractionation is less efficient when the initial water content is higher.

It is worth mentioning though that the pH abs H 2 O differences are much smaller than have been predicted by theoretical calculations [START_REF] Malevanets | Variation of droplet acidity during evaporation[END_REF].

Also it is important to note that it is not known whether the droplets formed in ESI are actually in their equilibrium. In previous studies it has been shown that some compounds may retain their liquid phase structures (charge location) due to kinetical trapping [START_REF] Seo | The impact of environment and resonance effects on the site of protonation of aminobenzoic acid derivatives[END_REF] meaning that the drying of the droplets in ESI is too fast for the droplets to reach the actual equilibrium. Therefore it is also likely that at least some of the droplets profiled in our work have not reached the chemical equilibria (including acid-base equilibrium). However calculations can only be made based on the assumption of reached equilibria.

The method used allows us to profile the droplets down to 0.5 μm in diameter, therefore indicating two mechanisms influencing the plume evolution. First, the drying of the droplets due to heated drying gas. In this process the more volatile mobile phase component evaporates faster, but the changes are expected to be similar for the two ESI modes.

Secondly, the fission of the charged nanodroplets from the parent droplets. This mechanism can have an effect on the composition of the remaining part of the parent droplet and is expected to influence ESI+ and ESI-mode differently. In ESI+ and ESI-ions of different type are removed from the parent droplets during ejection of nanodroplets, therefore facilitating pH abs H 2 O change in different directions. Each fission of one nanodroplets removes about 20% of the excess charge from the parent droplet [START_REF] Kebarle | On the mechanisms by which the charged droplets produced by electrospray lead to gas phase ions[END_REF][START_REF] Peschke | Features of the ESI mechanism that affect the observation of multiply charged noncovalent protein complexes and the determination of the association constant by the titration method[END_REF]. The general amount of excess charge can be estimated on the basis of pH change from bulk mobile phase to mobile phase in the needle tip. It is obvious that it changes from mobile phase to mobile phase, but based on the data obtained in this study it can be expected that the fraction of charge present as excess charge reaches several tens of per cent of the overall excess charge [START_REF] Enke | A predictive model for matrix and analyte effects in electrospray ionization of singly-charged ionic analytes[END_REF]. Therefore if the fission of nanodroplets would be a dominant process changing the composition of the parent droplets we would expect: (1) a significantly different These findings are very important from two aspects. First, for modelling ESI process it is obvious that desolvation due to drying is much more important than the fission of nanodroplets. This means that similar models for ESI+ and ESI-mode can theoretically be constructed and this could also serve as a validation criterion for such models.

Secondly, for practitioners it shows that qualitative findings related to the ESI process obtained on the ESI+ mode can almost directly be applied also in the ESI-mode. However the importance of desolvation gas is huge and therefore quantitative findings are transferable only within one set of gas parameters between ESI+ and ESI-.

Conclusions

Changes in organic content percentage, pH and droplet size are very similar for both ESI+ and ESI-indicating that in these means the two modes are similar. In all cases mobile phases became more acidic along the plume, but less than predicted by calculations. The results aid in understanding the conditions of where the ionization occurs better and enable to directly compare the two polarities used for ESI ionization efficiency measurements. This is very promising and our group is currently working on establishing solid quantitative link between the ionization 20 efficiencies in the two ionization modes, which would enable deciding whether ESI+ or ESI-is more promising for a given analyte.

pH abs H 2 O

 2 in the plume.

  the ratio of 80/20 and 50/50, and also for 80/20 mixture with aqueous solution with pH w w of 4.00. The corresponding pH abs H 2 O changes are listed in Table 2. The change of the droplet size was studied for acetonitrile/water 80/20 and 50/50 (v/v) mixtures with pH w w of 5.00, and also for 80/20 mixture with initial aqueous solution with pH w w of 4.00. The experimental setup profiling the spray plume consists of an excitation laser and two different optical detection systems mounted on a moving stage. It is coupled with a Single Quad 6100 mass spectrometer equipped with a modified Agilent Jet Stream ESI source (Agilent Technologies, Santa Clara, CA, USA) in order to allow the laser injection into the plume and the epifluorescence measurements. Used ESI parameters were: capillary voltage 3500/-3500 V, nebulizing gas pressure 15 psi, drying gas flow rate 7 L/min, drying gas temperature 300 °C, sheath gas flow rate 1 L/min and temperature 80 °C. Solutions were introduced in the ionization source at 50 µL min -1

  5 mm(divergence 1 mrad). The laser is injected through the objective using two reflecting protected aluminum flat mirrors (R > 90%). The laser beam is focused into the spray and the fluorescence is collected via an objective used in an epifluorescence configuration. Fluorescence spectra from ESI plume were recorded, point by point (pixel size 500 μm), by an ultra-compact spectrophotometer (B&WTek Inc., Newark, DE, USA). The plume was profiled for 15 mm starting from ESI needle tip, except for 50% MeCN solution where plume was profiled for 13 mm due to poor solubility of the fluorescent probe.The obtained raw data was processed using OriginPro 7.0 software. Profiles of pH and solvent composition in the ESI plume (Figures1a and 1b) were determined from calibrations in different solutions.For droplet size profiling (Figure1c), a Fraunhofer diffraction setup is implemented on the other side of the ESI source and the same excitation laser is used. A system of two cylindrical lenses is used to direct the diffracted light on the Charge Coupled Device (CCD) camera guppy F-080B/C FireWire (Allied Vision Technologies, Stadtroda, Germany). The first lens (f = 100 mm) allows the collection of the scattered light for a solid angle ranging from 𝛉 𝐦𝐢𝐧 = 𝟔. 𝟕° to 𝛉 𝐦𝐚𝐱 = 𝟏𝟕. 𝟕° with respect to the laser beam axis, whereas the second one (f = 150 mm) is used to focus the light on the CCD camera. The exposure time was set to 80 ms (with a time base of 20 µs) and 20 images are recorded for each point.

  fluorescent pH indicator 5(6)-carboxy-2',7'-dichlorofluoresceine (Sigma, ≥95%) and solvatochromic Nile Red (Invitrogen, Cergy, Pontoise, France) were used as fluorescent probes. Acetonitrile (J.T.Baker, Deventer, Netherlands, HPLC grade), MilliQ water (Millipore Advantage A10 MILLIPORE GmbH, Molsheim, France), formic acid (Fluka, 98%, Buchs, Switzerland), ammonium hydroxide (Lach:Ner, 25%, Czech Republic), dimethyl sulphoxide (Sigma, Steinheim, Germany), buffer pH 7.00 (Fluka, Buchs, Switzerland) were used. Water phase pH values ( pH w w ) were measured with Hanna Instruments pH211 Microprocessor pH Meter equipped with a 4 mmdiameter microelectrode (Pt // 3.5 mol/L KCl+AgCl).

  )-carboxy-2',7'dichlorofluoresceine were made in dimethyl sulphoxide. From the initial solution, 10 μM binary acetonitrile/water solutions were prepared, where the pH w w varied: 3.50, 4.00, 4.50, 5.03, 6.04, and 6.51. Water phase solutions were prepared by first making 0.1% formic acid solution and then adjusting pH by adding the ammonium hydroxide solution to the desired pH w w

Figure 1

 1 Figure 1 Change in (a) MeCN content in the middle of the ESI plume (y = 3 mm), (b) 𝐩𝐇 𝐚𝐛𝐬 𝐇 𝟐 𝐎 in the middle of the plume. pH in x = -1 mm is the 𝐩𝐇 𝐚𝐛𝐬 𝐇 𝟐 𝐎 of the initial mobile phase, (c) average droplet diameter in the spray plume versus the axial distance x from the emitter tip (average error is ±0.6 μm) (d) average droplet diameter versus the 𝐩𝐇 𝐚𝐛𝐬 𝐇 𝟐 𝐎 in ESI plume. The legend is the same for a, b, c and d. The droplet size was determined by Mie scattering measurements. The comparison of the

H 2 O

 2 along the plume is very similar in ESI+ and ESI-resulting in almost parallel evolution of lines (within one mobile phase) in Figure 1b. Therefore the pH abs H 2 O difference for ESI+ and ESI-generated by the electrochemical reaction in the needle tip remains essentially the same throughout the plume in spite of acetonitrile evaporation. Only in the case where buffer solution with pH w w of 4.00 was used, the aqueous phase of ESI+ becomes more acidic and ESI-becomes somewhat less acidic, making up the pH abs H 2 O difference of 0.53 units at the maximum profiling length.If electrochemical reactions would be of the same efficiency for all mobile phases and the organic solvent evaporation rates would be indistinguishable, as observed form Figure1a, we would expect that more acidic mobile phase obeys less pH change than the less acidic (also keeping in mind, that the buffering capacity for this studied mobile phase is much higher).Also it was observed that pH abs H 2 O change is larger for mobile phase containing 80% of acetonitrile than for mobile phase containing 50% of acetonitrile. The probable reasons are: (1) the profiled length is longer (15 mm versus 13 mm in the case of 50% MeCN) and (2) the change in the droplet size is larger (Figure1c and 1d). The first possibility may be ruled out, as the plateau of the pH change has already been achieved at x = 13 mm. The droplet size change, however, is in almost linear relation with pH abs H 2 O , see Figure 1d. It is observed, that the droplet size changes more for mobile phase with 80% of MeCN. MeCN evaporates more readily from solution with higher

pH abs H 2 O

 2 profile for ESI+ and ESI-along the plume and (2) more significant changes in the droplets pH abs H 2 O along the plume.

Table 1 .

 1 The acidity of mobile phases expressed as pH abs H 2 O values together with measurement uncertainties.

	Mobile phase a	𝐩𝐇 𝐚𝐛𝐬 𝐇 𝟐 𝐎	𝒖 𝐑𝐖
	𝐰 𝐰	5.50 80/20	8.59	0.01	0.14

b

𝒖 𝐂 c

MeCN/ 𝐩𝐇 a The aqueous phase is 0.1% formic acid solution titrated with 25% ammonia solution to the desired pH w w

Table 2 .

 2 Change of MeCN content and pH abs H mobile phase and mobile phase in the needle tip. The pH abs H 2 O change along the plume is visualized in Figure 1b. It can be observed that the pH abs H 2 O of the solution decreases steadily along

	Initial MeCN content	80%		80%		50%
	Initial pH w w	4.00		5.03		5.03
	Initial pH abs H 2 O	7.64		8.43		7.05
	ESI mode	ESI+	ESI-	ESI+	ESI-	ESI+	ESI-
	ΔMeCN content % (percentage point)	5.6%	5.7%	5.3%	7.4%	7.0%	7.5%
	pH abs H 2 O ( pH w w ) at x = 0 mm	7.47 (3.78)	7.81 (4.27)	8.20 (4.80)	8.49 (5.14)	6.83 (4.82)	7.19 (5.16)
	pH abs H 2 O ( pH w w ) at xmax	6.66 (3.21)	7.19 (3.91)	7.52 (4.28)	7.82 (4.73)	6.28 (4.54)	6.62 (4.88)
	H 2 O (Δ pH w w ) in ESI ΔpH abs plume	0.81 (0.57)	0.62 (0.36)	0.68 (0.52)	0.67 (0.41)	0.55 (0.28)	0.57 (0.28)
	The pH abs H

2 O in the ESI plume (y = 3 mm), xmax = 15 mm from the needle tip, except for 50% MeCN initial content, where xmax = 13 mm. 2 O at the ESI needle tip (x = 0 mm) is different (confirmed by t-test) from the pH abs H 2 O of the initial solution (

Table 2). The pH abs H 2 O change from 0.06 to 0.23 pH units is observed between the bulk

Acknowledgements

This work was supported by Personal Research Funding Project 34 from the Estonian Research Council, and by national scholarship program Kristjan Jaak, which is funded and managed by the Archimedes Foundation in collaboration with the Ministry of Education and Research. The research leading to these results has received funding from Agilent Technologies with an award through Agilent's Application and Core Technology University Research Program (Grant ID 2243). The work of AH and IL was supported by the Institutional funding project IUT20-14 from the Estonian Ministry of Education and Research.


Antonin Soleilhac

Franck Bertorelle

Philippe Dugourd

Marion Girod

Rodolphe Antoine
email: rodolphe.antoine@univ-lyon1.fr

Monitoring Methanol-induced protein unfolding by fluorescence anisotropy measurements of covalently labelled rhodamine probe

come

Monitoring methanol-induced protein unfolding by fluorescence anisotropy measurements of covalently labelled rhodamine probe

Introduction:

Protein conformational dynamics regulate protein function. Therefore, tools able to probe internal dynamics of proteins, in the picosecond to microsecond timescale are highly desirable. Fluorescence spectroscopy has become an established tool to investigate the dynamics and interactions of biomolecules. [START_REF] Lakowicz | Principles of Fluorescence Spectroscopy[END_REF] In particular, fluorescence spectroscopy is an important tool for the characterization of protein folding. [START_REF] Ladokhin | Encyclopedia of Analytical Chemistry[END_REF] As a possible route, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. [START_REF] Ha | [END_REF] However, the incorporation of two probes into a single protein for FRET studies can be difficult. Fluorescence anisotropy is an alternative route for the characterization of protein folding. 4 Measurements are based on the principle of the excitation of fluorophores by polarized light resulting in polarized emission. The polarized emission is influenced by a number of processes, including motions that occur within the lifetime of the excited fluorophore. Both intrinsic 5 and extrinsic 6 fluorophores can be used for fluorescence anisotropy measurements. Due to the comparable timescale of rotational diffusion of biopolymers and the fluorescence lifetime of many fluorophores, fluorescence anisotropy has been reported for numerous biological applications. Information on the shape and size of proteins can be obtained and then have been used to measure protein-protein interactions, 7 fluidity of membranes, 8 binding and conformational dynamics. 9 In this paper, we aim at probing methanol induced unfolding of a protein by fluorescence anisotropy measurements using an extrinsic fluorophore. A simple and reliable synthesis for labeling myoglobin protein with visible Rhodamine B chromophore is reported. Methanolinduced unfolding is revealed by the transition from constrained to free rotation of a covalently attached rhodamine B fluorophore.

Materials and Methods

Labeling Protocol

Myoglobin from equine skeletal muscle (Sigma-Aldrich) was labeled with Rhodamine B isothiocyanate (Sigma-Aldrich) according to a protocol slightly modified from Hungerford et al. 10 as follow: reagents were dissolved so that their concentration ratio is 1. Typically, 34 mg of myoglobin (Mb) and 1.1 mg of rhodamine B isothiocyanate (RhB) were dissolved in 10 mL of 0.1 M sodium bicarbonate buffer (pH 9) while stirring continuously for 2 hours at room temperature. The reaction associated to the labeling is given by: 11 In its native form, Mb is a globular protein having several available amine reactive groups. 12 The sequence chain of Mb and its 3D view are shown in Figure 1. Keeping the dye to protein ratio (D/P) around 1 is important to avoid any over-labeling of the protein; at most, only one dye is attached to the protein. Indeed, if the D/P ratio is greater than 1, it can lead to strong interactions between probe dye molecules grafted onto the same protein. 10 In order to remove any uncoupled RhB, the solution was then dialyzed using washed tubular cellulose membranes with a nominal molecular weight cut-off at 6000-8000 Da (ZelluTrans/Roth) against 2L of 10 mM Phosphate Buffered Saline (PBS) at pH 7 for one day. Several dialysis were performed until the absorption of RhB at 555 nm of the final supernatant was below the detection limit. Mixture of labeled protein and unlabeled protein was then recovered as is in PBS buffer as stock solution. Stock solution concentration is evaluated by absorption to 𝑐 𝑠𝑡𝑜𝑐𝑘 ~200 𝜇𝑀. Fluorescence measurements are performed after a 1:10 dilution. The final D/P ratio was determined using the molar extinction coefficients of Mb at 408 nm (188000 M -1 cm -1 ) 13 versus RhB one at 555 nm (106000 M -1 cm -1 ). 14

Fluorescence measurements

An Oxxius Slim 532-300 laser (Oxxius Inc., Santa Clara, CA, U.S.A.) was used for excitation. Fluorescence intensities were determined by the steady-state fluorescence spectra maximum recorded by an Ultracompact spectrophotometer Econic (B&WTek Inc., Newark, DE, U.S.A.) with a resolution of 1.5 nm via an optical fiber. 15,16 A polarizing beamsplitter cube and a halfwave plate were used to tune the linear polarity of the light on the excitation line. A linear broadband polarizer in the range 500 -720 nm was used for the collection. The alignment of the setup has been performed using the pure diffusion of the polarized light by a solution containing latex nanoparticles in suspension. For each fluorescence anisotropy measurements, the correction factor 𝐺 (defined below) was reevaluated. Vertically or horizontally excitations were tuned by rotating the halfwave plate by 45° according to the wanted configuration and vertically or horizontally emissions were collected by rotating the linear polarizer by 90° (see Figure 2). Fluorescence lifetimes of samples were measured by a HORIBA Scientific's hybrid spectrofluorometer Fluoromax, using a pulsed nanoled excitation at 372 nm and collecting the emitted light at 580 nm with a bandwidth of 5 nm. 17,18 Anisotropy Fluorescence -Principle When a fluorophore interacts with an electromagnetic wave, it can absorb a photon through the coupling of the electromagnetic field to its transition dipole moment. If the orientation of the electric filed vector of the excited light is parallel to the transition dipole moment of the fluorophore, the fluorophore can be excited efficiently. Hence, by exciting an ensemble of randomly oriented molecules with a linearly polarized light, only molecules that are aligned with the direction of the polarized excitation light are excited, so called photoselection. 19 If the fluorophore moves during the life span of its excited state (Rotational Brownian motion), the emitted light polarization will be modified. To quantify this loss of polarization, we define the fluorescence anisotropy 𝑟 as

𝑟 = 𝐼 𝑉𝑉 -𝐺𝐼 𝑉𝐻 𝐼 𝑉𝑉 +2𝐺𝐼 𝑉𝐻 (1)
where 𝐼 𝑉𝑉 corresponds to the fluorescence intensity of vertically polarized excitation and vertically polarized emission, 𝐼 𝑉𝐻 the vertically polarized excitation and horizontally polarized emission and 𝐺 is a correction factor defined by

𝐺 = 𝐼 𝐻𝑉 𝐼 𝐻𝐻 (2)
Assuming that rotational diffusion is the significant process that results a change of polarization, Perrin equation gives an expression of the fluorescence anisotropy

𝑟 = 𝑟 0 1+ 𝜏 𝜃 ⁄ (3)
where 𝑟 0 is the limiting anisotropy that would be measured in the absence of rotational diffusion -it refers to the existence of an angle between the absorption and the emission transition dipole moments -𝜏 is the lifetime of the fluorophore and 𝜃 the correlation time for the rotational process for a spherical objet that can by expressed from the Stokes-Einstein equation: 20

𝜃 = 𝜂𝑉 𝑘𝑇 ( 4
)
where 𝜂 is the viscosity of the solution, 𝑘 the Boltzmann constant, 𝑇 the temperature and 𝑉 the volume of the rotating object.

Results and discussion

Characterization of the sample

Absorption spectra of Mb, RhB and of the solution of the complex (RhB-labeled Mb) right after the final dialysis in 10 mM Phosphate Buffer Solution (PBS) are shown in Figure 3. The characteristic Soret band at 408 nm denotes the presence of the heme group on the Mb. The existence of the RhB absorption pick at 555nm for the complex solution right after the final dialysis clearly indicates that the labeling occurs efficiently. We can estimate by ratiometry of the respective absorbance at 555 and 408 nm that the D/P ratio is approximately 1/3.

Mass Spectrometry (MS) measurements of

RhB-labeled Mb in water (PBS) are shown in Figure S1a. Characteristic peaks of holo-Mb in its native form with a low Charge State Density are clearly predominant (9 + , 8 + and 7 + ), along with one-dye labeled proteins also visible in their native form with the same Charge State Density with a typical ratio consistent with the D/P ratio estimated by absorbance ratiometry.

Circular Dichroism (CD) measurements of RhB-labeled Mb in water (PBS) are displayed in Figure S2. Both mass spectra and CD spectra confirm that the presence of a covalently attached dye onto the protein doesn't affect the structure of the protein, regarding either its Charge State Density (cf. mass spectrometry) or its secondary structure (cf. CD spectra).

Fluorescence lifetimes of the different samples are reported in Table 1.

Measurements show that attached dye fluorescence lifetimes are similar to those of dye alone in the same solution. Thus the protein backbone does not seem to affect the lifetime of the rhodamine dye. This might indicate a dye labeling at the surface of the protein which does not significantly affect the excitation lifetime. Furthermore, measured fluorescence lifetime decreases when increasing the water proportion in the solution revealing that the RhB fluorescence is quenched by water.

RhB alone in a PBS displays a fluorescence anisotropy of 𝑟 = 0.046 ± 0.005 whereas for the RhB-labeled protein 𝑟 = 0.199 ± 0.005 . If we assume that Mb displays a globular shape in its native form in PBS, we can estimate that the system radius of gyration is equal to 𝑟 𝑔 = 1.6 ± 0.1 𝑛𝑚 considering a limiting anisotropy 𝑟 0 = 0.3 , and a viscosity 𝜂 = 8.95 × 10 -4 𝑃𝑎. 𝑠. This value is consistent with the literature. 12,21 Parameters' influence on fluorescence anisotropy.

According to expressions ( 3) and ( 4), fluorescence anisotropy is affected by the following main physical parameters: (i) Temperature is a key parameter regarding Brownian motion. For high temperature, objects motion is greater resulting in a loss of anisotropy. (ii) The size of the moving objects also greatly influence the anisotropy. The bigger an object is, the slower its rotation will be, resulting in a gain of anisotropy. (iii) High viscosity tends to slow down the rotation motion inducing an anisotropy increase. (iv) It is also clear that the fluorescence lifetime plays a role in the depolarization process. For short lifetime, the molecule reemits light rapidly so that it doesn't rotate enough to induce a great depolarization. We can calculate the predicted evolution of RhB emission anisotropy regarding the size of the rotating system (ie the radius of gyration) for a given temperature in a water/MeOH mixture in the range 0 -30% of methanol. 22 Results are given in Figure 4 taking into account viscosity and lifetime modifications due to different solvent conditions. It is shown that for a given size, fluorescence anisotropy of a system tends to increase with the methanol proportion. Indeed, the anisotropy increase induced by the change of viscosity 22 counterbalances the anisotropy loss due to the augmentation of the fluorophore lifetime.

Probing unfolding of myoglobin by fluorescence anisotropy measurements

Alcohols are well-known solvents that highly weaken hydrophobic interaction leading to strong denaturation of proteins. 23 24, 25 In order to probe protein dynamic events due to methanol (MeOH) addition, fluorescence anisotropy of RhB-labeled Mb has been measured as a function of methanol proportion at room temperature.

Results shown in Figure 5 clearly indicate a depolarization of fluorescence emission when increasing the methanol proportion.

According to the previous discussion, a loss of anisotropy may be explained by a reduction of the size of the object. However, this is inconsistent with Ion Mobility Spectrometry measurements performed on Mb. Indeed, for each charge state, several are observed, and a global augmentation of the cross-section occurs when the solvent is changed from water to water/methanol. 26 MS measurements on Mb in different solvent conditions (Figure S1b) show that the average Charge State Density of Mb tends to increase with methanol which is consistent with a partial unfolding of the protein. Moreover, the signature of apo-Mb induced by the liberation of the noncovalent Heme group is observed in MS spectra and is a signature of a partial opening of the protein. Furthermore, CD analysis reveals a slight denaturation from alpha helix to unordered structure when increasing the methanol proportion (see Table S1). All these considerations are favorable to a partial unfolding of the protein leading to an augmentation of its radius of gyration. Thus, the drop in anisotropy can only be explained as an effect of the protein unfolding. The decrease is attributable to a global unfolding which allows the fluorophore to rotate independently and freely from the protein backbone. Several models have been developed in the literature to discuss processes where independent motions of the transition dipole moment (protein chain or covalently attached chromophore) are considered. [27][28][29] This phenomenon has already been observed on depolarization tryptophan UV emission. Indeed, the decrease was attributable to a great extent of unfolding which releases the tryptophan residue from the hydrophobic core of the protein

% MeOH

Anisotropy allowing the indole ring to rotate independently of the protein. 4 However, the anisotropy value of the system stays significantly bigger than the free fluorophore's one alone in solution because the fluorophore is still covalently attached to the protein (See Figure S3 for more details). The circular dichroism spectra reported for Mb have signatures that are typical for helical structure. 26 One may argue that structuration of Mb in -helix upon addition of methanol avoids a strong interaction of the rhodamine B dye with the protein peptidic skeleton.

Conclusion

A simple and reliable synthesis for labeling a protein with a visible chromophore is reported. Optical characterization of the system is performed through absorption spectrometry in the visible range, mass spectrometry, circular dichroism, fluorescence lifetime and fluorescence anisotropy measurements. It is shown that at most, one rhodamine B isothiocyanate molecule is grafted to myoglobin via an amine group to avoid any possible interactions within the molecules onto the same protein and no effect of the grafting can be detected regarding the main structure of the protein. Moreover, unfolding events of the protein induced by organic solvent like methanol can be probed by fluorescence anisotropy measurements. Such events are revealed by the transition from "constrained" to "free" rotation of the attached fluorophore. The present study suggests that it should be possible, with fluorescent techniques using extrinsic fluorophores to obtain information in kinetic studies of protein folding and conformational changes.

Figure 1 (

 1 Figure 1 (a) Sequence Chain of Myoglobin. Amine groups are noted in red and α-helix regions are highlighted in yellow (b) 3D view of Myoglobin, reprinted from Protein Data Bank (ref: 5CMV). Figure 2 Schematic view of the experimental setup to measure fluorescence anisotropy 𝑟. The excitation and detection polarization are here vertical. The λ/2 plate and the linear polarizer are mounted on rotation plates.

Figure 3 :

 3 Figure 3: Absorption spectra of (black) Mb, (red) RhB and (blue)RhB-labeled Mb in 10 mM PBS buffer.

Figure 4 :

 4 Figure 4: Calculated fluorescence anisotropy regarding the radius of gyration of a system. Inset: Anisotropy for a given radius of gyration (𝑟 𝑔 = 1.6 𝑛𝑚) regarding the methanol proportion. Values of 𝜏 and 𝜂 have been reevaluated as follow: For 0% of MeOH, 𝜏 = 1.73 ns, 𝜂 = 8.95 x 10 -4 Pa.s For 10% of MeOH, 𝜏 = 1.88 ns, 𝜂 = 11.55 x 10 -4 Pa.s For 20% of MeOH, 𝜏 = 1.97 ns, 𝜂 = 13.92 x 10 -4 Pa.s For 30% of MeOH, 𝜏 = 2.03 ns, 𝜂 = 15.40 x 10 -4 Pa.s

Figure 5 :

 5 Figure 5: Fluorescence anisotropy of RhB-labeled Mb in water/MeOH mixture.

Table 1 :

 1

		Fluorescence Lifetime (ns)
	Vol. % of MeOH	Free Rhodamine B isothiocyanate	Rhodamine B isothiocyanate labeled on Myoglobin
	0	1.75	1.73
	10	1.87	1.88
	20	1.95	1.98
	30	2.02	2.04

Fluorescence lifetime of free and RhB-labeled Mb in water/MeOH mixture in 10 mM PBS buffer.

Supporting Information

Mass spectrum of RhB-labeled Mb in Water (PBS) and Mb in different solvent conditions. Circular Dichroism spectra of Mb and RhB-labeled Mb in Water (PBS). Average values of the secondary structure for RhBlabeled Mb in different solvent conditions.

Protocol and absorption spectra of RhBlabeled Mb in water (PBS) with and without the presence of methanol.


Manuel Atencia

Jérôme David

Jérôme Euzenat

Liliana Ibanescu

Nathalie Pernelle

Fatiha Saïs

Élodie Thiéblin

Cassia Trojahn

Discovering Expressive Rules for Complex Ontology Matching and Data Interlinking

Introduction

Ontology matching and data interlinking as distinct tasks aim at facilitating the interoperability between different knowledge bases. Although the field has fully developed in the last years, most ontology matching works still focus on generating simple correspondences (e.g., Author ≡ W riter). These correspondences are however insufficient to fully cover the different types of heterogeneity between knowledge bases and complex correspondences are required (e.g., LRIM ember ≡ Researcher ∃belongsT oLab. {LRI}). Few approaches have been proposed for generating complex alignments, focusing on correspondence patterns or exploiting common instances between the ontologies. Similarly, unsupervised data interlinking approaches (which do not require labelled samples) have recently been developed. One approach consists in discovering linking rules on unlabelled data, such as simple keys [START_REF] Symeonidou | Sakey: Scalable almost key discovery in rdf data[END_REF] (e.g., {lastN ame, lab}) or conditional keys [START_REF] Symeonidou | VICKEY: mining conditional keys on knowledge bases[END_REF] (e.g., {lastN ame} under the condition c = Researcher ∃lab.{LRI}). Results have shown that the more expressive the rules are, the higher the recall is. However naive approaches cannot be applied on large datasets. Existing approaches presuppose either that the data conform to the same ontology [START_REF] Symeonidou | Sakey: Scalable almost key discovery in rdf data[END_REF] or that all possible pairs of properties be examined [START_REF] Atencia | Data interlinking through robust linkkey extraction[END_REF]. Complementary, link keys are a set of pairs of properties that identify the instances of two classes of two RDF datasets [START_REF] Atencia | Data interlinking through robust linkkey extraction[END_REF] (e.g., { creator, auteur , title, titre } linkkey Book, Livre , expresses that instances of the Book class which have the same values for properties creator and title as an instance of the Livre class has for auteur and titre are the same). Such, link keys may be directly extracted without the need for an alignment.

for establishing complex correspondences [START_REF] Atencia | Data interlinking through robust linkkey extraction[END_REF]. Then, the key discovery tools VICKEY [START_REF] Symeonidou | VICKEY: mining conditional keys on knowledge bases[END_REF] and LinkEx are applied for the discovery of simple keys, conditional keys, and link keys from the instances of O 1 and O 2 , exploiting the complex correspondences as input (as a way of reducing the key search space) (2). The keys are then applied in the data interlinking task, which can also benefit from the complex correspondences (as a way of extending the sets of instances to be compared) [START_REF] Symeonidou | VICKEY: mining conditional keys on knowledge bases[END_REF]. Finally, as CANARD considers shared instances, the matching is iterated by considering the detected identity links. We plan to evaluate the approach to verify, on the one hand, whether the use of complex correspondences allows to improve the results of data interlinking. On the other hand, thanks to the use of the detected identity links, it would also be reasonable to expect improvements in ontology matching results. Experiments will be run on DBpedia and YAGO, covering different domains such as people, organizations, and locations, as there exists reference entity links or these datasets.

Fig. 1 .

 1 Fig. 1. Workflow of ontology matching and data interlinking enhanced by key discovery.

Acknowledgement. This work is supported by the CNRS Blanc project RegleX-LD.