Daiki Asakawa
email: d.asakawa@aist.go.jp

Akio Miyazato

Frédéric Rosu

Valérie Gabelica

Influence of the Metals and Ligands in Dinuclear Complexes on Phosphopeptide Sequencing by Electron Transfer Dissociation Tandem Mass Spectrometry

Keywords: Radical site, Phosphorylation site, Density functional theory, Reduction

Phosphorylation is one of the most important protein modification, and electron transfer dissociation tandem mass spectrometry (ETD-MS/MS) is a potentially useful method for sequencing of phosphopeptide, including determination of phosphorylation site. Notably, ETD-MS/MS typically provide useful information when precursor contains more than three positive charges and has not yet closed as the method for large-scale phosphopeptide analysis, due to the difficulty of the production for acidic phopeptides having more than three positive charges. To increase the charge state of phosphopeptides, we have used the dinuclear metal complexes, which selectively bound to phosphate group in phosphopeptide with addition of positive charge(s). The dinuclear copper, zinc and gallium complexes have been tested and the type of metal present in the complex strongly affected to the affinity of phosphorylated compound and their ETD fragmentation. The dinulcear copper complex interact weakly with phosphate group and ETD induced peptide fragmentation is largely suppressed by the presence of Cu 2+ , which worked as an electron trap. The dinulcear gallium complex strongly bound to phosphate group. However, the ligand binding to gallium acted as an electron trap and the presence of dinulcear gallium complex in precursor for ETD-MS/MS hampers the sequencing of phosphopeptide, as in the case of dinulcear copper complexes. In contrast, dinulcear zinc complexes efficiently bind to phosphopeptides with increase in the charge state, facilitating phosphopeptide sequencing by ETD-MS/MS. The fragmentation of ligand and peptide backbone in dinulcear zincphosphopeptide complex are competitively induced by ETD. These processes are influenced by ligand structure and the detailed ETD fragmentation pathway were investigated using density functional theory calculations.

INTRODUCTION

The reversible phosphorylation of proteins is a common covalent protein modification, [START_REF] Cohen | The Regulation of Protein Function by Multisite Phosphorylation-A 25 Year Update[END_REF][START_REF] Hunter | Signaling-2000 and Beyond[END_REF] and electrospray ionization (ESI)-based tandem mass spectrometry (MS/MS) is currently the dominant method for the analysis of protein phosphorylation. In particular, collision-induced dissociation (CID) has been widely used to analyze the peptide sequence by MS/MS. In the CID of phosphorylated peptides, the loss of the phosphate groups (80 and 98 Da) can be used as a specific marker for phosphorylated peptide identification. [START_REF] Boersema | Phosphopeptide Fragmentation and Analysis by Mass Spectrometry[END_REF][START_REF] Palumbo | Tandem Mass Spectrometry Strategies for Phosphoproteome Analysis[END_REF] Therefore, the screening of phosphorylated peptides can be achieved by CID-MS/MS. However, the determination of the phosphorylated sites in peptides is hindered by the substantial loss of the phosphate groups because most of the fragment ions are observed in a non-phosphorylated form in CID-MS/MS spectra. [START_REF] Boersema | Phosphopeptide Fragmentation and Analysis by Mass Spectrometry[END_REF][START_REF] Asakawa | Mass Spectrometric Characterization of Phosphorylated Peptides using MALDI In-Source Decay via Redox Reactions[END_REF][START_REF] Asakawa | De Novo Sequencing of Tryptic Phosphopeptides using Matrix-Assisted Laser Desorption/Ionization Based Tandem Mass Spectrometry with Hydrogen Atom Attachment[END_REF] Fragmentation methods involving the electron association of multiply charged peptides, such as electron capture dissociation (ECD) [START_REF] Zubarev | Electron Capture Dissociation of Multiply Charged Protein Cations. A Nonergodic Process[END_REF] and electron transfer dissociation (ETD) [START_REF] Syka | Peptide and Protein Sequence Analysis by Electron Transfer Dissociation Mass Spectrometry[END_REF] , have been used as alternative methods to CID. [START_REF] Kelleher | Peer Reviewed: Top-Down Proteomics[END_REF][START_REF] Coon | Collisions or Electrons? Protein Sequence Analysis in the 21st Century[END_REF][START_REF] Zhou | Advancements in Top-down Proteomics[END_REF] Because the ECD/ETD of phosphorylated peptides results in fragment ions arising from the fragmentation of the peptide backbone without losing phosphoric acid, the determination of the phosphorylation site is possible. Regarding the ECD/ETD mechanism, electron attachment/transfer occurs competitively at positively charged sites [START_REF] Zubarev | Electron Capture Dissociation for Structural Characterization of Multiply Charged Protein Cations[END_REF] and at the π* antibonding orbital of peptide bond [START_REF] Sobczyk | Coulomb-Assisted Dissociative Electron Attachment: Application to a Model Peptide[END_REF][START_REF] Syrstad | Toward a General Mechanism of Electron Capture Dissociation[END_REF][START_REF] Asakawa | N-Cα Bond Cleavage of Zinc-Polyhistidine Complexes in Electron Transfer Dissociation Mediated by Zwitterion Formation: Experimental Evidence and Theoretical Analysis of the Utah-Washington Model[END_REF] in multiply charged peptides, leading to N-Cα bond cleavage through aminoketyl radical intermediates. Although one of the most promising applications of ECD/ETD is phosphopeptide sequencing, it has not yet become the method of choice for large-scale phosphopeptide analysis because of the low fragmentation efficiency for tryptic phosphopeptides. [START_REF] Asakawa | High-Confidence Sequencing of Phosphopeptides by Electron Transfer Dissociation Mass Spectrometry Using Dinuclear Zinc(II) Complex[END_REF] Employing ions with a higher charge state as the precursors for ECD/ETD is a way to dramatically improve the fragmentation efficiency by increasing the yield of reactive radical species, and thereby improve the sequence coverage. [START_REF] Rožman | Charge State Dependent Top-Down Characterisation Using Electron Transfer Dissociation Rapid[END_REF][START_REF] Good | Performance Characteristics of Electron Transfer Dissociation Mass Spectrometry[END_REF][START_REF] Frese | Improved Peptide Identification by Targeted Fragmentation Using CID, HCD and ETD on an LTQ-Orbitrap Velos[END_REF] Here, we investigated how to increase the charge state of precursor ions to improve the quality of ETD mass spectra. In particular, the complexation of peptides with metal ions has been demonstrated to increase the charge state of peptides and various metal cations have been used as the cationization adductive for ETD-MS/MS experiments. [START_REF] Flick | Electron Capture Dissociation of Trivalent Metal Ion-Peptide Complexes[END_REF][START_REF] Asakawa | Influence of Metal-Peptide Complexation on Fragmentation and Inter-Fragment Hydrogen Migration in Electron Transfer Dissociation[END_REF][START_REF] Asakawa | Electron Transfer Dissociation Mass Spectrometry of Peptides Containing Free Cysteine Using Group XII Metals as a Charge Carrier[END_REF][START_REF] Chen | Dissociation of trivalent metal ion (Al(3) (+) , Ga(3) (+) , In(3) (+) and Rh(3) (+) )-peptide complexes under electron capture dissociation conditions[END_REF][START_REF] Commodore | Effects of Acidic Peptide Size and Sequence on Trivalent Praseodymium Adduction and Electron Transfer Dissociation Mass Spectrometry[END_REF] With regards to the ETD processes, the metal-peptide complex undergoes either N-Cα bond cleavage or metal cation reduction, according to the electrochemical properties of metal cations in the precursor.

The metal cation having closed shell, such as monovalent alkali metal cations, divalent alkali earth and group XII metal cations, and trivalent group XIII metal cations are barely reduced by ETD-MS/MS condition. [START_REF] Iavarone | Effects of Charge State and Cationizing Agent on the Electron Capture Dissociation of a Peptide[END_REF][START_REF] Fung | Electron Capture Dissociation of Peptides Metalated with Alkaline-Earth Metal Ions[END_REF][START_REF] Chen | Transition Metal Ions: Charge Carriers that Mediate the Electron Capture Dissociation Pathways of Peptides[END_REF][START_REF] Bogdanov | Electron Capture Dissociation Studies of the Fragmentation Patterns of Doubly Protonated and Mixed Protonated-Sodiated Peptoids[END_REF] In consequence, the use of metal cation having closed shell promotes the N-Cα bond cleavage and provides more informative MS/MS mass spectra compared to those obtained from protonated precursors with lower charge states. [START_REF] Asakawa | Influence of Metal-Peptide Complexation on Fragmentation and Inter-Fragment Hydrogen Migration in Electron Transfer Dissociation[END_REF][START_REF] Asakawa | Electron Transfer Dissociation Mass Spectrometry of Peptides Containing Free Cysteine Using Group XII Metals as a Charge Carrier[END_REF][START_REF] Commodore | The Effects of Trivalent Lanthanide Cationization on the Electron Transfer Dissociation of Acidic Fibrinopeptide B and its Analogs[END_REF] In contrast, transition metal cations with a partially filled d orbital shell, such as Co 2+ , Ni 2+ , and Cu 2+ , are reduced by ETD and the fragmentation is mainly induced by the substantial excitation attributable to the reduction energy of the metal cation in the complex. [START_REF] Asakawa | Difference of Electron Capture and Transfer Dissociation Mass Spectrometry on Ni 2+ -, Cu 2+ -and Zn 2+ -Polyhistidine Complexes in the Absence of Remote Protons[END_REF] Although Cd 2+ and Hg 2+ are closed shelled metal cation, Cd 2+ -and Hg 2+ -peptide complexes undergoes metal cation reduction by ETD, probably due to complex electron energy level of Cd 2+ and Hg 2+ . [START_REF] Asakawa | Electron Transfer Dissociation Mass Spectrometry of Peptides Containing Free Cysteine Using Group XII Metals as a Charge Carrier[END_REF][START_REF] Chen | Formation of Peptide Radical Cations (M + •) in Electron Capture Dissociation of Peptides Adducted with Group IIB Metal Ions[END_REF] The results suggest that the period 5, 6 and 7 metal cations were susceptible to undergo the reduction by ETD. Therefore, alkali metals, alkaline earth metals, Al 3+ , Zn 2+ and Ga 3+ are suggested to be suitable adductive for peptide sequencing by ETD-MS/MS. However, the yield of metal-peptide complex is usually low when the free metal cation was used. [START_REF] Asakawa | Influence of Metal-Peptide Complexation on Fragmentation and Inter-Fragment Hydrogen Migration in Electron Transfer Dissociation[END_REF][START_REF] Asakawa | Electron Transfer Dissociation Mass Spectrometry of Peptides Containing Free Cysteine Using Group XII Metals as a Charge Carrier[END_REF][START_REF] Asakawa | Difference of Electron Capture and Transfer Dissociation Mass Spectrometry on Ni 2+ -, Cu 2+ -and Zn 2+ -Polyhistidine Complexes in the Absence of Remote Protons[END_REF] To increase the yield of metal-phosphopeptide complex, we have recently used a dinuclear zinc complex, (Zn2L1) 3+ (L1 = alkoxide form of 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-ol), also known as the phosphate capture molecule "phos-tag", [START_REF] Kinoshita | Recognition of Phosphate Monoester Dianion by An Alkoxide-Bridged Dinuclear Zinc(II) Complex[END_REF][START_REF] Kinoshita | Novel Immobilized Zinc(II) Affinity Chromatography for Phosphopeptides and Phosphorylated Proteins[END_REF][START_REF] Kinoshita | Phosphate-Binding Tag, A New Tool to Visualize Phosphorylated Proteins[END_REF] as an ionizing reagent. [START_REF] Asakawa | High-Confidence Sequencing of Phosphopeptides by Electron Transfer Dissociation Mass Spectrometry Using Dinuclear Zinc(II) Complex[END_REF] The molecular structure of (Zn2L1) 3+ is shown in Scheme 1a. Because (Zn2L1) 3+ selectively binds with phosphorylated peptides with the addition of a positive charge per phosphate group, (Zn2L1) 3+ -aided ESI improves the ionization yield of phosphopeptides present in the protein digest, [START_REF] Asakawa | High-Confidence Sequencing of Phosphopeptides by Electron Transfer Dissociation Mass Spectrometry Using Dinuclear Zinc(II) Complex[END_REF] and allowed us to sequence tryptic phosphopeptide by ETD-MS/MS. However, (Zn2L1) 3+ -aided ETD-MS/MS had one disadvantage: the fragmentation of (Zn2L1) 3+ was also induced by ETD and competed with phosphopeptide backbone fragmentation. Therefore, the development of a new metal complex to replace (Zn2L1) 3+ is necessary to suppress the ligand dissociation and to preferentially form the fragments arising from N-Cα bond cleavage. Similarly to (Zn2L1) 3+ , dinuclear metal complexes with (2,6-bis[(N,N′-bis[2picolyl]amino)methyl]-4-tertbutylphenol, L2), such as (Co2L2) 3+ , (Cu2L2) 3+ , (Zn2L2) 3+ , (Ga2L2) 5+ , and (In2L2) 5+ have been also tested as phosphate capture molecules. [START_REF] Svane | Overcoming the Instability of Gaseous Peptide Phosphate Ester Groups by Dimetal Protection[END_REF][START_REF] Svane | Charge Inversion of Phospholipids by Dimetal Complexes for Positive Ion-Mode Electrospray Ionization Mass Spectrometry Analysis[END_REF][START_REF] Svane | Effect of Metals in Biomimetic Dimetal Complexes on Affinity and Gas-Phase Protection of Phosphate Esters[END_REF][START_REF] Svane | The Selectivity of Water-Based Pyrophosphate Recognition is Tuned by Metal Substitution in Dimetallic Receptors[END_REF] The molecular structure of (Me n+ )2(L2) -1 , where Me n+ is Cu 2+ , Zn 2+ or Ga 3+ , is shown in Scheme 1b. In this study, we tested the applicability of the dimetallic complexes (Cu2L1) 3+ , (Cu2L2) 3+ , (Zn2L2) 3+ , and (Ga2L2) 5+ for ESIbased ETD-MS 2 analysis of phosphopeptides and compared the results with those from the (Zn2L1) 3+aided method. As described above, Zn 2+ and Ga 3+ are suitable adductive for peptide sequencing by ETD-MS/MS. In consequence, we used Zn 2+ and Ga 3+ as the nuclears in the complex and complexes with Cu 2+ was used as the reference. First, we evaluate the efficiency for the binding of phosphorylated serine (pS) to the dimetallic complexes from ESI mass spectra. We then used the dimetallic complexes as additives for the ETD-MS/MS analysis of tryptic phosphopeptides, T18p (NVPLpYK), T19p (HLADLpSK), and T43p (VNQIGpTLSESIK), which have been used as models in previous studies. [START_REF] Asakawa | High-Confidence Sequencing of Phosphopeptides by Electron Transfer Dissociation Mass Spectrometry Using Dinuclear Zinc(II) Complex[END_REF] Although ESI of these phosphopeptides produced singly and doubly protonated molecules, the triply protonated phosphopeptides were absent. The precise location of the phosphorylation site could not be determined because of the low sequence coverage when doubly protonated molecules were used as the precursors for ETD-MS/MS. [START_REF] Asakawa | High-Confidence Sequencing of Phosphopeptides by Electron Transfer Dissociation Mass Spectrometry Using Dinuclear Zinc(II) Complex[END_REF] In contrast, the use of zinc complexes increased in the charge state of phosphopeptides in ESI-MS and facilitated the phosphopeptide sequencing by subsequent ETD-MS/MS analysis. To address the general mechanism for the ETD of the dimetal-ligand-phosphopeptide complexes, the details of the ETD fragmentation pathway were investigated using density functional theory (DFT) calculations.

EXPERIMENTS

Materials

Synthetic phosphopeptides designed to mimic the fragments produced by the tryptic digestion of yeast enolases, T18p, T19p, and T43p, were purchased from CS Bio Co., Ltd. (Shanghai, China).

The phosphorylated Nα-acetylated serine-lysine dipeptide (Ac-pSK) was purchased from GenScript Inc. (NJ, USA). The detailed information for the model phosphopeptides are summarized in Table 1.

Paraformaldehyde, sodium triacetoxyborohydride, 2-pyridinecarboxyaldehyde, 1,3diamino-2-propanol, copper (II) chloride, zinc (II) chloride, and gallium (III) perchlorate were supplied by Sigma-Aldrich Inc. (MO, USA). Bis(2-pyridylmethyl)amine and p-cresol were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).

All solvents used were of high-performance liquid chromatography grade, except for water, which was purified by a Milli-Q ® purification system (Millipore; Billerica, MA, USA). The conductivity of the water was 18.2 MΩ/cm.

Synthesis of ligand molecules

Ligand L1 was synthesized from 1,3-diamino-2-propanol and 2-pyridinecarboxaldehyde by dehydration condensation in methanol. To facilitate the reaction, sodium triacetoxyborohydride was added. The final concentrations of 1,3-diamino-2-propanol, 2-pyridinecarboxaldehyde, and sodium triacetoxyborohydride were approximately 0.15, 0.75, and 0.84 M, respectively. The reaction was performed for 5 days at room temperature. Ligand L2 was synthesized from p-cresol, p-formaldehyde, and bis(2-pyridylmethy)amine in ethanol using a literature method. [START_REF] Ghiladi | Dinuclear Iron(III)-Metal(II) Complexes as Structual Core Models for Purple Acid Phosphatases[END_REF] The final concentration of pcresol, p-formaldehyde, and bis(2-pyridylmethy)amine were approximately 0.35, 0.8, and 0.8 M, respectively, and the reaction was performed under reflux conditions over 3 days. Synthesized ligands L1 and L2 were purified by column chromatography with silica gel and recrystallization, respectively.

To obtain dinuclear metal complexes, the synthesized ligands, L1 and L2, were dissolved in methanol at 0.15 M, and the obtained solutions were mixed with aqueous solutions of the corresponding metal salt, i.e., CuCl2, ZnCl2, or Ga(ClO4)3. The mixing molar ratio of ligand/metal was approximately 1/2. The complexes were obtained by re-crystallization.

Measurement of Binding Affinity between pS and (Me n+ )2(Lx -1 ) by ESI-MS

ESI-MS was used to investigate the trend of binding affinities between pS and (Me n+ )2(Lx -1 ),

where Me n+ is Zn 2+ , Cu 2+ , or Ga 3+ , and Lx is L1 or L2. The abundances of free (Me n+ )2(Lx -1 ) and (Me n+ )2(Lx -1 )(pS) complexes were estimated by positive ion ESI-MS using an Orbitrap Exactive mass spectrometer (ThermoFisher, Bremen, Germany). The dinuclear complexes, (Me n+ )2(Lx -1 ), were dissolved in water/methanol (1/1, v/v) at a concentration of 20 μM. The complexation was studied in solutions containing 10 µM (Me n+ )2(Lx -1 )(pS) and variable concentrations of pS (from 0.5 to 100 μM).

The final concentration of the dinuclear complex was adjusted to 10 μM for all experiments. The values for the abundance of (Me n+ )2(Lx -1 ) and (Me n+ )2(Lx -1 )(pS) were estimated from the peak areas of the corresponding signals observed in the ESI mass spectra and the mass balance equation on the total concentration of [(Me n+ )2(Lx -1 ).

ETD-MS/MS

The analyte peptides were dissolved in water/methanol (1/1, v/v) at a concentration of 10 μM.

To produce the dinuclear complex-phosphopeptide by ESI, (Cu2Lx) 3+ , (Zn2Lx) 3+ , or (Ga2L2) 5+ were added to the peptide solution at concentrations of 100, 40, and 20 μM, respectively. ESI mass spectra were acquired using a 9.4-T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (SolariX FT, Bruker, Germany). The analyte solution was directly infused into the mass spectrometer using ESI. The ion accumulation time, ion cooling time, and time-of-flight values were set to 0.5 s, 20 ms, and 7 ms, respectively. For the ETD-MS/MS experiments, the precursor ions were mass-selected in the quadrupole filter and then reacted with the fluoranthene radical anion. The times for reagent accumulation and the ion/ion reaction were set to 150 and 25 ms, respectively. Total ESI-MS and subsequent ETD-MS/MS mass spectra were obtained by the accumulation of 20 and 100 single mass spectra, respectively.

Computational details

All electronic structure calculations were performed with Gaussian 16. 40 The geometries for the zinc trihistidine complexes were optimized by DFT calculations using the MN15 41 hybrid functional and double-zeta valence polarized basis sets, i.e., LanL2DZ for the metal atoms and 6-31G(d) for the C, H, O, N, and P atoms. To establish the fragmentation energies, transition state (TS) geometries were also optimized at the MN15/LanL2DZ/6-31G(d) level of theory and confirmed by vibrational frequency analysis to have one imaginary frequency. The relationship between the transition state and the reactants, as well as the intermediates, was checked by intrinsic reaction coordinate analysis [START_REF] Fukui | The Path of Chemical Reactions-the IRC Approach[END_REF] starting from the transition state conformation. Single-point energies of the local energy minima and transition state geometries were calculated using the MN15 functional with the 6-31++G(2d,p) basis set. Excited electronic states were calculated using a time-dependent DFT method [START_REF] Stratmann | An Efficient Implementation of Time-Dependent Density-Functional Theory for the Calculation of Excitation Energies of Large Molecules[END_REF] with the MN15 functional and the 6-31++G(2d,p) basis set.

Notation

In the present study, Zubarev's notation was adopted for peptide fragment ions. [START_REF] Zubarev | Reactions of Polypeptide Ions with Electrons in the Gas Phase[END_REF]

RESULTS AND DISCUSSION

Formation Efficiency of Dinuclear Metal-pS Complexes

First, we focused on the binding properties of dimetallic complexes to pS, which were estimated from the ESI mass spectra. In this study, five dimetallic complexes, (Zn2L1) 3+ , (Zn2L2) 3+ , (Cu2L1) 3+ , (Cu2L2) 3+ and (Ga2L2) 5+ were tested. Because the utility of the (Zn2L1) 2+ -aided ETD-MS/MS for phosphopeptide sequencing were described by previous report, [START_REF] Asakawa | High-Confidence Sequencing of Phosphopeptides by Electron Transfer Dissociation Mass Spectrometry Using Dinuclear Zinc(II) Complex[END_REF] we first discussed on the complex formation between (Zn2L1) 3+ and pS. Figure 1a shows the ESI mass spectra of the mixture of (Zn2L1) 3+ and pS in different mixing ratios.

As described in experimental section, (Zn2L1) 3+ was synthesized from [L1 + H] and ZnCl2.

Thereby, Cl -would be present as counter ion of (Zn2L1) 3+ , and (Zn2L1) 3+ was detected as the complex with Cl -, [Zn2L1 + Cl] 2+ , when the concentration of pS was lower than that of (Zn2L1) 3+ (upper panel of Figure 1a). In addition, was suggested to be originated from the (Zn2L1) 3+ and pS complex, as shown in Scheme 2.

Scheme 2. The proposed structure of ions observed in ESI mass spectra of (Zn2L1) 3+ and pS mixtures, as shown in Figure 1a.

Next, we focused on the complexation efficiency of (Zn2L1) 3+ binding to pS. To estimate the complexation efficiency, we performed ESI-MS of a (Zn2L1) 3+ and pS mixture at a range of pS concentrations from 0.5 to 40 μM. The concentration of (Zn2L1) 3+ was set to 10 μM for all experiments.

Figure 1b shows the relationship between the signal intensity ratio of (Zn2L1) 3+ with and without pS, and the total pS concentration between 0.5 and 40 μM. The values of the signal ratio were calculated from the sum of the intensities of all species assigned in Figure 1a. The yield of the complex between (Zn2L1) 3+ and pS increased with an increasing concentration of pS and 60 % of (Zn2L1) 3+ was bound to pS, when the concentration of (Zn2L1) 3+ and pS were 10 μM.

As in the case of (Zn2L1) 3+ , (Zn2L2) 3+ selectively bound to the phosphate group of pS, producing [Zn2L2 + pS -H] 2+ . The relationship between the amount of (Zn2L1) 3+ with and without pS at different concentrations of pS is shown in Figure 1c. The comparison of Figures 1b and1c indicated the yield of complex formation between (Zn2L2) 3+ and pS was slightly lower than that of (Zn2L1) 3+ and pS. In consequence, the (Zn2L1) 3+ would capture the phosphate group more efficiently. Next, we investigated dinuclear copper complexes, (Cu2L1) 3+ and (Cu2L2) 3+ , for their use as phosphate capture molecules. Figure 2a shows the ESI mass spectra for the mixture of 10 μM (Cu2L1) 3+ and pS with mixing ratios, [(Cu2L1) 3+ ]/[pS], of 1 and 6. As in the case of (Zn2L1) 3+ , the ESI mass spectra of the mixture of (Cu2L1) 3+ and pS contained chloride and pS adduct forms, [Cu2L1+Cl] 2+

and [Cu2L1 + pS -H] 2+ . In contrast, the fragment ions [CuL1 + H] 2+ and [Cu2L1 + H2PO4] 2+ were absent. In terms of relative affinities, although (Zn2L1) 3+ was mostly bound to pS when 40 μM of pS was added to the (Zn2L1) 3+ solution (Figure 1a), the chloride adduct of (Cu2L1) 3+ was detected as an intense signal at 40 μM of added pS (Figure 2a). (Cu2L2) 3+ showed similar behavior to (Cu2L1) 3+ (data not shown). Next, the formation of the (Cu2L1) 3+ -pS and (Cu2L2) 3+ -pS complexes were determined using the same procedure as for the (Zn2L1) 3+ -pS complex (Figures 2b and2c). The comparison of Figures 1 and2 indicate that (Cu2L1) 3+ and (Cu2L2) 3+ have lower phosphate capturing ability than (Zn2L1) 3+ and (Zn2L2) 3+ . Thus, the phosphate capturing ability of dinuclear metal complex is strongly affected by the type of metal ion. Next, (Ga2L2) 5+ was used as a phosphate capture molecule. Because Ga is a trivalent metal ion, the use of (Ga2L2) 5+ is expected to increase the charge state of the phosphorylated peptides. Figure 3a shows the ESI mass spectra for the mixture of 10 μM (Ga2L2) 5+ and pS with mixing ratios,

[pS]/[(Ga2L2) 5+ ], of 0.2 and 1. When [pS]/[(Ga2L2) 5+ ] was 0.2, (Ga2L2) 5+ was detected as [Ga2L2 + O] 3+ , [Ga2L2 + O + OH] 2+ and [Ga2L2 + O + ClO4] 2+ . The source of ClO4 -is the Ga(ClO4)3 used for the synthesis of (Ga2L2) 5+ . According to the previous reports, (Ga2L2) 5+ is estimated to be present as (Ga2L2)(OH)2(ClO4)3(H2O)2 in solid state. [START_REF] Svane | The Selectivity of Water-Based Pyrophosphate Recognition is Tuned by Metal Substitution in Dimetallic Receptors[END_REF] Because OH -and ClO4 -were presented as the adduct ions in ESI mass spectra, the counter ions of (Ga2L2) 5+ in solution are suggested as OH -and ClO4 The addition of pS contributes to increasing the signal intensity of the (Ga2L2) 5+ -pS complex, [Ga2L2 + pS -3H] 2+ (lower panel of Figure 3a). (Ga2L2) 5+ would coordinate to a deprotonated phosphate group and a carboxylic acid group to form [Ga2L2 + pS -3H] 2+ . Figure 3b shows the relationship between the amount of (Ga2L2) 5+ for a total pS concentration in the range of 0.5-40 μM.

The amount of (Ga2L2) 3+ with and without pS was estimated from the peak areas of

[Ga2L2 + O] 3+ , [Ga2L2 + O + OH] 2+ , [Ga2L2 + O + ClO4] 2+
, and [Ga2L2 + pS -3H] 2+ . (Ga2L2) 5+ was mostly bound to pS when 20 μM of pS was added to the 10 μM (Ga2L2) 5+ solution. The comparison of Figures 123indicates that (Ga2L2) 5+ has the highest binding ability for pS among the complexes tested in this study.

ETD-MS 2 of the Dinuclear Metal-Phosphopeptide Complexes

We also investigated the suitability of the complexes for phosphopeptide sequencing by ETD-MS/MS. The phosphopeptide T18p was used as the model peptide and triply-charged dinuclear metal-T18p complexes were used as the precursor ions for ETD-MS/MS experiments. Figure 4 shows the ETD-MS/MS spectra of [Zn2L1 + T18p] 3+ , [Zn2L2 + T18p] 3+ , [Cu2L1 + T18p] 3+ , and [Ga2L2 + (T18p -2H)] 3+ . The (Zn2L1) 3+ complex was used in our previous work as an additive to increase the charge state of phosphorylated peptides, facilitating phosphopeptide sequencing by ETD-MS/MS. [START_REF] Asakawa | High-Confidence Sequencing of Phosphopeptides by Electron Transfer Dissociation Mass Spectrometry Using Dinuclear Zinc(II) Complex[END_REF] As described in the introduction, the ETD of the (Zn2L1) 3+ -phosphopeptide complex produces an intense signal of the fragment ion arising from a 92-Da loss, which corresponds to ligand degradation. The presence of an intense signal corresponding to the fragment ion because of the 92-Da loss hinders phosphopeptide sequencing. As in previous reports, [START_REF] Asakawa | High-Confidence Sequencing of Phosphopeptides by Electron Transfer Dissociation Mass Spectrometry Using Dinuclear Zinc(II) Complex[END_REF] Supporting Information, Scheme S3. In summary, the use of (Ga2L2) 5+ for ETD MS/MS preferentially provide 92-Da loss and did not induce peptide backbone fragmentation. Therefore, the (Ga2L2) 5+ -aided ETD are not suitable for phosphopeptide sequencing, although (Ga2L2) 5+ strongly bind to phosphate group.

The Utility of (Zn2L2) 3+ for Phosphopeptide Sequencing by ETD-MS/MS

As described in the previous section, the use of (Zn2L2) 3+ potentially facilitates phosphopeptide sequencing by ETD-MS/MS. Next, we used, T19p, and T43p as tryptic phosphopeptide models. These phosphopeptides have been used as models in previous studies, but the precise location of the phosphorylation site could not be determined because of the low sequence coverage when doubly protonated molecules were used as the precursors for ETD-MS/MS. [START_REF] Asakawa | High-Confidence Sequencing of Phosphopeptides by Electron Transfer Dissociation Mass Spectrometry Using Dinuclear Zinc(II) Complex[END_REF] Figure 7 shows the (Zn2L2) 3+ -aided ETD-MS/MS mass spectra when triply charged (Zn2L2) 3+ -phosphopeptide complexes were used as the precursors. (Zn2L2) 3+ -aided ETD-MS/MS provided almost full sequence coverage, including information concerning the phosphorylation site. For comparison with the previously reported (Zn2L1) 3+ -aided ETD-MS/MS method, the change from (Zn2L2) 3+ to (Zn2L1) 3+

significantly suppressed the yield of the fragment arising from the 92-Da loss. In addition, the efficiency of N-Cα bond cleavage was improved, indicating that (Zn2L2) 3+ -aided ETD-MS/MS is a useful method for the sequencing of phosphopeptides.

CONCLUSION

The dinuclear metal complexes (Zn2L1) 3+ , (Zn2L2) 3+ , (Cu2L1) 3+ , (Cu2L2) 3+ , and (Ga2L2) 5+

were investigated for use as additives for the analysis of phosphopeptides by ESI-based ETD-MS/MS.

Although all dinuclear metal complexes selectively bound to phosphate compounds, their affinity is dependent on the type of metal present in the complex. The order of binding affinity for pS is (Ga2L2) 5+ > (Zn2L1) 3+ ≳ (Zn2L2) 3+ > (Cu2L1) 3+ > (Cu2L2) 3+ .

The type of metal ion in the complex also strongly influenced the ETD fragmentation.

Regarding the ETD of the complexes with copper, Cu 2+ acted as an electron trap, and peptide fragmentation was largely suppressed. In contrast, (Ga2L2) 5+ strongly bound to pS and phosphopeptides, whereas the ETD of the (Ga2L2) 5+ -phosphopeptide complex mainly led to C6H6N• loss. Therefore, the (Cu2L1) 3+ , (Cu2L2) 3+ , and (Ga2L2) 5+ -aided ETD-MS/MS could not be used for phosphopeptide sequencing.

The zinc dinuclear complexes, (Zn2L1) 3+ and (Zn2L2) 3+ , efficiently bound to the phosphorylated compounds. In particular, the use of (Zn2L2) 3+ facilitated N-Cα bond cleavage by ETD-MS/MS compared with the (Zn2L1) 3+ -aided method. The yield of the fragments arising from N-Cα bond cleavage reflects the number of aminoketyl radical intermediates, as predicted by TD-DFT calculations. The computational results indicate that the (Zn2L2) 3+ -phosphopeptide complex can efficiently produce aminoketyl radical intermediates by ETD, facilitating phosphopeptide sequencing.

To apply (Zn2L2) 3+ -aided ETD for large-scale phosphopeptide analysis, the method should be combined with liquid chromatography mass spectrometry (LC-MS) system. Although the (Zn2L2) 3+phosphopeptide complex would not be stable during chromatographic separation process, the post-column addition of (Zn2L2) 3+ can be produced the complex. The further investigation for (Zn2L2) 3+aided ETD combined with LCMS would promote wider application of this method.

Scheme 1 .

 1 Scheme 1. Structure of dinuclear complexes (a) (Me n+ )2(L1 -1 ) and (b) (Me n+ )2(L2 -1 ), where Me n+ is Zn 2+ , Cu 2+ , or Ga 3+ .

  According to this notation, homolytic N-Cα bond cleavage yields the radical c• and z• fragments, and the addition of a hydrogen atom to the c• or z• fragments produces a c' or z' fragment, respectively. The abstraction of a hydrogen atom from the c• or z• fragments produces a c or z fragment, respectively.

Figure 1 .

 1 Figure 1. (a) ESI mass spectra of (Zn2L1) 3+ and pS mixtures with different concentration ratios.

Figure 2 .

 2 Figure 2. (a) ESI mass spectra of (Cu2L1) 3+ and pS mixtures with different concentration ratios.

Figure 3 .

 3 Figure 3. (a) ESI mass spectra of (Ga2L2) 5+ and pS mixtures with different concentration ratios.

  the ETD of [Zn2L1 + (Ac-pSK -H)] 2+ showed the fragment ion arising from the 92-Da loss as an intense signal, in addition of formation of the fragments due to N-Cα bond cleavage. In contrast, the ETD-MS/MS mass spectrum of [Zn2L2 + T18p] 3+ showed a more intense signal corresponding to fragment ions arising from N-Cα bond cleavage, and a lower intensity of the fragment ion arising from the 92-Da loss, as compared to when (Zn2L1) 3+ was used.As shown in Figure4c, the ETD of [Cu2L1 + T18p] 3+ , generated charge reduced products, [Cu2L1 + T18p] 2+ , and [Cu2L1 + T18p] + . In addition, (CuL1) + and [T18p + Cu] + were observed as the fragment ion. Importantly, the [T18p + Cu] + contained monovalent copper atom, indicating that ETD induced reduction of Cu 2+ . Because the binding energy between (Cu2L1) 3+ and T18p was decreased by reduction of Cu 2+ in the complex, (CuL1) + and [T18p + Cu] + would be formed by ETD-MS/MS. This result is good agreement with previously reported ETD-MS/MS of Cu 2+ -peptide complex[START_REF] Asakawa | Difference of Electron Capture and Transfer Dissociation Mass Spectrometry on Ni 2+ -, Cu 2+ -and Zn 2+ -Polyhistidine Complexes in the Absence of Remote Protons[END_REF] and the presence of Cu 2+ in the precursor ion suppress the N-Cα bond cleavage by ETD-MS/MS.Regarding the Figure4d, ETD of [Ga2L2 + (T18p -2H)] 3+ selectively induced C6H6N• losses, leading to the [Ga2L2 + (T18p -2H -C6H6N)] 2+ and [Ga2L2 + (T18p -2H -2C6H6N)] + . The C6H6N• loss was induced the electron association to the pyridine ring in the complex, and the N-Cα bond cleavage is completely suppressed by the presence of (Ga2L2) 5+ in the precursor.

Figure 4 .

 4 Figure 4. ETD-MS/MS mass spectra of the doubly charged complexes: (a) [Zn2L1 + T18p] 3+ , (b) [Zn2L2 + T18p] 3+ , (c) [Cu2L1 + T18p] 3+ and (d) [Ga2L2 + (T18p -2H)] 3+ . Asterisks and black

Figure 5 .

 5 Figure 5. ETD-MS/MS mass spectra of the doubly charged complexes: (a) [Zn2L1 + (Ac-pSK -H)] 2+ , (b) [Zn2L2 + (Ac-pSK -H)] 2+ , (c) [Cu2L1 + (Ac-pSK -H)] 2+ , and (d) [Ga2L2 + (Ac-pSK -3H)] 2+ . Asterisks and black squares indicate the precursor ions and charge reduced products,

Figure 6 .

 6 Figure 6. Optimized conformations and electronic state diagrams for vertical electron

Figure

  Figure5dshows ETD-MS/MS mass spectrum of [Ga2L2 + (Ac-pSK -3H)] 2+ , which

Figure 7 .

 7 Figure 7. ETD-MS/MS mass spectra of triply charged (Zn2L2) 3+ -phosphopeptide complexes. The

Table 1 . Monoisotopic Mass (Mm), Sequence, and Composition of the Analyte Phosphopeptides Used

 1

	Analyte	Mm	Sequence	Composition
	pS	185.08	pS	C3H8NO6P
	Ac-pSK	346.11	Ac-pSK	C11H23N3O8P
	T18p	812.39	NVPLpYK	C35H57O12N8P
	T19p	862.40	HLADLpSK	C34H59O14N10P
	T43p	1367.68	VNQIGpTLSESIK C55H98O23N15P

  Ga3+ . The proton transfer reaction between hydroxides produced the intermediate complex, IM, consisted by [Ga2L2 + O] 3+ and H2O. The corresponding transition state was TS in Scheme S1, which is 30 kJ/mol less stable than reactant. The complete dissociation energy of [Ga2L2 + O] 3+ and H2O from [Ga2L2 + 2OH] 3+ was 66 kJ/mol. Because the internal energies of the ions produced by ESI with standard parameter sets is estimated to be around 180 kJ/mol,[START_REF] Gabelica | Internal energy and fragmentation of ions produced in electrospray sources[END_REF] the proposed formation pathway of [Ga2L2 + O] 3+ is feasible.

		-. In
	consequence, [Ga2L2 + O] 3+ might be formed from [Ga2L2 + 2OH] 3+ during ESI process by chemical
	reaction (1) and proposed structure of [Ga2L2+O] 3+ was shown in Scheme 3.
	[Ga2L2 + 2OH] 3+ → [Ga2L2 + O] 3+ + H2O	(1)
	To investigate the reaction behaviors of [Ga2L2 + 2OH] 3+ in details, we calculated optimized

geometries by DFT with MN15/6-31++G(2d,p)//MN15/LanL2DZ/6-31G(d) level (Supporting information, Scheme S1). In the lowest energy geometry of [Ga2L2 + 2OH] 3+ , a hydroxide was strongly coordinated by both Ga 3+ and another hydroxide weakly bound to a

Table 2 . Reaction energies of ETD-induced fragmentation, as shown in Scheme 4. The relative energies (kJ/mol) were obtained from MN15/6-31++G(2d,p)//MN15/LanL2DZ/6-31G(d) calculations.

 2

	Reaction

ACKNOWLEDGMENTS

DA acknowledge Issey Osaka, for allowing to use FT-ICR MS instrument. This work was supported by JSPS KAKENHI grant number 17K14508. The computations of molecular structures in this work were supported by Research Center for Computational Science, Okazaki and Center for Computational Sciences. The experiments were partly supported by Nanotechnology Platform Program of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and the Plateforme de Biophysico-Chimie Structurale of the IECB, France.


Valérie Gabelica
email: v.gabelica@iecb.u-bordeaux.fr

Sandrine Livet

Frederic Rosu

Frédéric Rosu

Optimizing Native Ion Mobility Q-TOF in Helium and Nitrogen for Very Fragile Noncovalent Structures

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

Native mass spectrometry (native MS) involves measuring ions that have preserved a memory of the non-covalent interactions that were present in solution. [START_REF] Schalley | Supramolecular chemistry goes gas phase: the mass spectrometric examination of non-covalent interactions in host-guest chemistry and molecular recognition[END_REF][START_REF] Loo | Studying non-covalent protein complexes by electrospray ionization mass spectrometry[END_REF][START_REF] Smith | The observation of non-covalent interactions in solution by electrospray ionization mass spectrometry: promise, pitfalls and prognosis[END_REF][START_REF] Schalley | Mass Spectrometry and Gas-Phase Chemistry of Non-Covalent Complexes[END_REF] Intramolecular non-covalent interactions are responsible for the folding into specific secondary and tertiary structures, and intermolecular noncovalent interactions are responsible for the assembly of several molecules into complexes, also called "quaternary structures". The principles underlying native MS are common to mass spectrometry of intact biologically relevant molecules (proteins, nucleic acids, sugars) and of synthetic architectures [START_REF] Schalley | Supramolecular chemistry goes gas phase: the mass spectrometric examination of non-covalent interactions in host-guest chemistry and molecular recognition[END_REF] (artificial foldamers or supramolecular complexes). The main difference lies in the solution conditions in which the samples are dissolved for the analysis: biologically relevant complexes are studied in an electrospray-compatible buffer that best mimics the native cell environment in order to draw conclusions relevant to biology, [START_REF] Leney | Native Mass Spectrometry: What is in the Name?[END_REF] whereas artificial architectures can be studied in any medium of interest.

The experimentalist should always ensure minimal perturbation of the non-covalent interactions to be detected in the gas phase, so that the measurements provide information on the structures that were originally present in the injected solution. For a mass analysis (detection of the complexes), this means that the intermolecular non-covalent interactions must be preserved from the source to the mass analyzer. For an ion mobility (IM) analysis (characterization of the shapes via the friction with a gas when dragged by an electric field [START_REF] Kanu | Ion mobility-mass spectrometry[END_REF][START_REF] Clemmer | Ion mobility measurements and their applications to clusters of biomolecules[END_REF] ), this means that the intramolecular non-covalent interactions must be preserved from the source to the mobility cell. [START_REF] Wyttenbach | Intermolecular interactions in biomolecular systems examined by mass spectrometry[END_REF] Here we focused on the drift tube ion mobility instrument from Agilent (the 6560 IMS Q-TOF), [START_REF] May | Conformational ordering of biomolecules in the gas phase: nitrogen collision cross sections measured on a prototype high resolution drift tube ion mobility-mass spectrometer[END_REF] which like the Waters Vion IMS-QTof, the Tofwerk IMS-TOF or the Bruker timsTOF has an ESI-IM-Q-TOF configuration (as opposed to the Waters Synapt™ instruments which have an ESI-Q-IM-TOF configuration). In this configuration, both rupture of intramolecular interactions before the IM cell and rupture of the intermolecular interactions between the IM cell and the mass analyzer can affect the interpretation of ion mobility results. Pre-IM activation causes a gas-phase unfolding that could be falsely attributed to a solution unfolding. [START_REF] Gabelica | Fundamentals of Ion Mobility Spectrometry[END_REF][START_REF] Clemmer | Characterizing the Conformationome: Toward a Structural Understanding of the Proteome[END_REF] Also, in IM calibration procedures, it is also extremely important that the calibrant ions are produced with the same distribution of collision cross sections in the calibrating instrument and in the calibrated instrument. [START_REF] Bush | Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology[END_REF] For this reason, native proteins were found to be poorer calibrants than denatured ones. [START_REF] Sun | Protein Structural Studies by Traveling Wave Ion Mobility Spectrometry: A Critical Look at Electrospray Sources and Calibration Issues[END_REF] On the other hand, pre-IM activation can be exploited to study the gas-phase unfolding pathways, in so called collision-induced unfolding (CIU) experiments, [START_REF] Zhong | Collisional and Coulombic Unfolding of Gas-Phase Proteins: High Correlation to Their Domain Structures in Solution[END_REF] which are the IM analogue of collision-induced dissociation (CID) experiments in MS. Finally, post-IM fragmentation is often to be avoided as well, because if the arrival time distribution of a given m/z range is actually the arrival time distribution of the parent ion that traveled in the IM and later led to the product ion having this particular m/z, IM could be misinterpreted.

The degree to which non-covalent interactions can be altered in the gas phase depends on the internal energy of the ions, on the number of degrees of freedom on which this internal energy is redistributed, on the number and strength of the interactions involved, on the strength of Coulomb repulsion. Thus, complexes of smaller size and of higher charge density are more sensitive to ion activation. Here we present how we optimized the Agilent 6560 IMS Q-TOF. We used bovine ubiqutin 7+ to minimize pre-IMS unfolding and the DNA G-quadruplex [(dG 4 T 4 G 4 ) 2 •(NH 4 + ) 3 -8H] 5-, which undergoes ammonia loss at low internal energy, [START_REF] Balthasart | Ammonium ion binding to DNA G-quadruplexes: do electrospray mass spectra faithfully reflect the solution-phase species?[END_REF] to further minimize post-IMS fragmentation. Ubiquitin has frequently been used to assess IM instrument softness, because the IM profile of charge states 6+ to 8+ is extremely sensitive to internal energy effects, whether in the source, [START_REF] Li | Influence of solvent composition and capillary temperature on the conformations of electrosprayed ions: unfolding of compact ubiquitin colformers from pseudonative and denatured solutions[END_REF] in the transfer, [START_REF] Koeniger | Transfer of Structural Elements from Compact to Extended States in Unsolvated Ubiquitin[END_REF] or in the pre-IM trapping region. [START_REF] Myung | Structural Transitions of Electrosprayed Ubiquitin Ions Stored in an Ion Trap over ~10 ms to 30 s[END_REF][START_REF] Wyttenbach | Structural stability from solution to the gas phase: native solution structure of ubiquitin survives analysis in a solvent-free ion mobility-mass spectrometry environment[END_REF] The solution composition also plays a role on the detected gas-phase conformations. [START_REF] Li | Influence of solvent composition and capillary temperature on the conformations of electrosprayed ions: unfolding of compact ubiquitin colformers from pseudonative and denatured solutions[END_REF][START_REF] Shi | Solution dependence of the collisional activation of ubiquitin [M + 7H](7+) ions[END_REF] The native state of ubiquitin 7+ has a collision cross section of ~1000 Ų in helium (~1300 Ų in nitrogen). In contrast with a recent report on the same instrument, [START_REF] May | Conformational landscapes of ubiquitin, cytochrome c, and myoglobin: Uniform field ion mobility measurements in helium and nitrogen drift gas[END_REF] we could obtain predominantly the native state on ubiquitin 7+ , both in nitrogen and in helium drift tube conditions. For the G-quadruplex, we found that fragmentation mainly occurred after the IM when using the default instrumental settings, and when lowering the voltage gradient one had to trade-off softness for reduced IM resolution. We will describe here the rationale for optimizing the instrument for native MS of analytes bearing extremely energysensitive non-covalent interactions.

Experimental

Bovine ubiquitin, >98% purity (SDS-PAGE) was purchased from Sigma (St Quentin Fallavier, France). The oligodeoxynucleotide strand dGGGGTTTTGGGG was purchased from Eurogentec (Seraing, Belgium). The G-quadruplex was prepared by mixing 100 µM single strands in 150 mM aqueous ammonium acetate, and waiting at least 24 h. Ammonium acetate 5 M solution (molecular biology) was from Fluka. Water was nuclease-free from Ambion™ (ThermoFischer Scientific, Illkirch, France). Acetic acid glacial, formic acid 99%, and methanol absolute were from Biosolve (Dieuze, France). Sulfolane 99% was from Aldrich (St Quentin Fallavier, France).

All experiments were run on an Agilent 6560 IMS-Q-TOF, [START_REF] May | Conformational ordering of biomolecules in the gas phase: nitrogen collision cross sections measured on a prototype high resolution drift tube ion mobility-mass spectrometer[END_REF] equipped with the alternate gas kit, which regulates the drift tube pressure with a flow controller based on continuous readings with a capacitance gauge. The pumping system was modified in-house by using the default scroll pump for the rear of the instrument only, and connecting an ECODRY 40 Plus pump (Leybold France SAS, Les Ulis, France) to the source region. To ensure appropriate pressure differentials for collision cross section determination, the pressures were set to 3.76 Torr in the drift tube and 3.47 Torr in the trapping funnel before the flow controller was turned on. This resulted, with the flow controller on, in pressures 3.89 Torr in the drift tube and 3.63 Torr in the trapping funnel. The differential (0.26 Torr) ensured that only the intended gas (helium or nitrogen) is present in the drift tube, despite using atmospheric pressure ionization. The acquisition software version was B.07.00 build 7.00.7008. The IM data processing software IM-MS browser B.07.02 build 7.02.210.0 was used to calculate collision cross section values of well-defined peaks, and to export arrival time distributions. All graphs were constructed using SigmaPlot 12.5. For the CIU plots, because exported arrival time distributions lack data points in regions where there is no intensity; each arrival time distribution was smoothed using a negative exponential, sampling 0.02 and polynomial order 1, to produce evenly spaced data (200 intervals).

For ubiquitin in the positive mode, optimization of the pre-IMS zone was done with a 0.75 µM solution in 99% H 2 O and 1% acetic acid, like in the Bleiholder group did for the soft tuning of trapped IMS, [START_REF] Liu | On the structural denaturation of biological analytes in trapped ion mobility spectrometry -mass spectrometry[END_REF] on the 7+ ion. Table 1 lists the parameter setpoints optimized by the manufacturer (on the Agilent tunemix ions) at the end of the installation of our instrument. We also list the parameters described by May et al. on their ubiquitin study, and the full list of parameters further optimized in the present study. The effects of changing each parameter will be described in the remainder of the paper. To optimize the post-IMS zone for softness, we used the fragile bimolecular G-quadruplex [(dGGGGTTTTGGGG) 2 •(NH 4 + ) 3 -8H] 5-ion, which readily loses ammonia upon collisional activation. [START_REF] Balthasart | Ammonium ion binding to DNA G-quadruplexes: do electrospray mass spectra faithfully reflect the solution-phase species?[END_REF] It is the most collision-sensitive specific nucleic acid complex we know of, and frequently use it for instrument tuning. [START_REF] Balthasart | Ammonium ion binding to DNA G-quadruplexes: do electrospray mass spectra faithfully reflect the solution-phase species?[END_REF] The optimized pre-IMS parameters were first transposed to the negative mode from the ubiquitin optimized ones, by changing all signs. Then, the voltages gradients behind the IMS were lowered. MS/MS mode was off (collision energy = 0 V). Table 2 lists three sets of the various parameters that can be tuned: the default parameters at installation (which give the best transmission and mobility resolution), the parameters optimized for softness for the very fragile G-quadruplex, while still preserving most of the ion signal and a decent mobility separation (although with lower resolving power, see results and discussion), and a compromise that works for most moderately fragile complexes. Further examples are given in the results and discussion.

Table 2: Tuning parameters (negative mode) in the post-IMS region. The "optimized" and "compromise" parameters are transposable to the positive mode by reversing the signs.

Parameter Installation by

Agilent, suitable for intramolecular folding studies

Compromise for moderately fragile noncovalent complexes

Optimized on very fragile Gquadruplex

IM drift tube: Drift tube exit -250 V -210 V -210 V IM rear funnel: Rear funnel entrance -240 V -200 V -200 V IM rear funnel: Rear funnel RF 150 V p-p 180 V p-p 180 V p-p IM rear funnel: Rear Funnel Exit -43 V -35 V -26 V IM rear funnel: IM Hex Entrance -41 V -32 V -24 V IM rear funnel: IM Hex Delta -9 V -3 V -2 V Optics 1: Oct Entrance Lens -32 V -27 V -21 V Optics 1: Oct 1 DC -31.3 V -25 V -20 V Optics 1: Lens 1 -29.4 V -23 V -19 V Optics 1: Lens 2 disabled disabled disabled Quad: Quad DC -27.8 V -21 V -18 V Quad: PostFilter DC -27.8 V -21 V -17 V Cell: gas flow 22 psi 20 psi 20 psi Cell: Cell Entrance -26.8 V -20 V -16 V Cell: Hex DC -25.8 V -20 V -16 V Cell: Hex Delta 9 V 3 V 3 V Cell: Hex2 DC -16.6 V -14.6 V -12 V Cell: Hex2 DV 3 V 1.5 V 1 V Optics 2: Hex3 DC -13.2 V -12.9 V -11 V Extractor: Ion Focus -10 V -10 V -10 V

Results and discussion

Tuning is a multiparametric and iterative process. To improve softness, the general reasoning is to reduce the ion acceleration by electric fields to reduce the laboratory frame collision energy between the ion and the background gas. However, there is often a compromise between softness and ion transmission. Also, too much softness can mean insufficient desolvation or declustering. Finally, the actual effect of a given voltage difference depends on the gas pressure and flows. [START_REF] Schmidt | Influence of pressure in the first pumping stage on analyte desolvation and fragmentation in nano-ESI MS[END_REF][START_REF] Gabelica | Internal energy and fragmentation of ions produced in electrospray sources[END_REF] We will show the effect of each type of parameter either side of our final optimized values to illustrate what happens when departing from this optimum. It must be understood however that there may exist different sets of parameter which could constitute another optimum. We don't claim we have reached the global optimum, but we have reached one that suits our applications on native MS of ~10 kDa complexes and very fragile complexes, including for performing collision-induced unfolding experiments.

Tuning the instrument pre-IM region for softness

In the pre-IM region, we examined twenty different parameters. Figure 1 depicts the optimized DC voltage gradient from the end of the glass capillary to the IM entrance. Supporting information Figure S1 compares visually the DC gradient in the default instrument tuning (which we presume is similar to that used by May et al. [START_REF] May | Conformational landscapes of ubiquitin, cytochrome c, and myoglobin: Uniform field ion mobility measurements in helium and nitrogen drift gas[END_REF] unless otherwise specified in Table 1) and in the optimized tuning for the drift tube operated in helium. As detailed below, the main difference lies in the voltage gradients in the ion trapping region.

The voltage called "fragmentor", applied at the end of the glass capillary, was already known to influence softness. However, the tuning of the other parameters was less intuitive, and we found one voltage difference (between the trap entrance and the trap entrance grid) that unexpectedly had a large influence on the results. All parameters are explained in detail below. We focus here on describing the tuning for softness with the drift tube in helium, which was more sensitive than in nitrogen, and will mention any differences found in nitrogen. The parameters are listed in Table 1. The same number of scans were summed in all cases. The drift tube was operated with V = 390 V. The peak at ~25 ms corresponds to the native form (CCS He = 981 Ų; standard deviation was 7 Ų from 10 independent measurements). The optimum value is in bold. Effects of A) voltages on the whole trapping region (see inset of Figure 1), the values written being the trap entrance grid low-trap entrance-trap exit-trap exit grid 1 low-trap exit grid 2 low; B) trap funnel delta; C) HP funnel RF and front funnel RF, both set at the same value; D) trap release time; E) trap fill time; F) HP funnel delta and fragmentor voltage; G) electrospray nebulizer pressure; H) dry gas flow; I) dry gas temperature effect, in order of recording. Note that after lowering the temperatures for a while, the system takes a very long time to recover its initial condition.

DC level of the trapping region.

All DC voltages are floating on the IM entrance, which is thus defined here as zero volts. The trapping funnel is located before the IM entrance. The trapping funnel has an hourglass shape, with an entrance, an exit, and the trapping region delimited by grids. Further towards the source we find the high pressure funnel (which has entrance and exit voltages), and the "fragmentor", which accelerates ions from the glass capillary towards the HP funnel. As shown in Figure 1 and Table 1, on the data acquisition software for some voltages one enters absolute values, and for others one enters voltages differences ("delta"s). The trap funnel exit is optimum at 10 V in all cases.

The voltage levels of the trapping region of the trapping funnel (in the 70-volts range on Figure 1) will influence the acceleration energy of ions released from the trap and injected in the drift tube.

In Figure 2A, we show that this "injection energy" influences the softness. In helium, when the trap DC voltages are in the 90-volts or 100-volts range, such as in typical installation parameters for nitrogen, the conditions are less soft and the peak of the native conformation at ~25 ms disappears to the profit of more extended (longer drift time) conformations. Too low trap DC voltages are also detrimental, because the IM peaks are shifted to longer times. The reason is that the ions travel slower to the IM: this delay contributes to the dead time t 0 (time spent outside the drift region), and the conditions are detrimental to the resolving power. In helium, the optimum trap DC level is thus in the 70-vols range. In nitrogen, the activation effects are less acute (see supporting Figure S2A), and the 90V-range is optimum, because in the 70V-range the ions drift too slowly towards the IM. The influence of other voltages in the trapping region will be discussed in the section on CIU. The parameters listed in Table 1 are the softest ones that do not compromise ion trapping efficiency or IM resolution.

Funnel DC gradients. Continuing towards the source, we have the trap funnel entrance, which voltage is defined by the trap funnel exit voltage plus the trap funnel delta. Figure 2B shows the influence of the trap funnel delta. At high values (200 V is the maximum allowed), the conditions become slightly activating, and one loses signal intensity. But counterintuitively, at low voltages (100 V) the conditions are also more activating, and the signal is lower. A possible explanation is that if the ions move too slowly between the funnel entrance through the hourglass to the trapping region, they wander around longer before entering in the trapping region and have time to be activated by the RF and change conformation. Our results therefore suggest that ion trapping in the first part of the funnel can occur at too low voltages. The optimum front funnel delta voltage was set to 140 V in helium (160 V in nitrogen, in line with the trap level which is also 20 V higher, see Figure S2B). In contrast, the high-pressure funnel delta shows no such effects, and any value of HP funnel delta between 60 V and 160 V gives similar results (Figure 2C). A possible explanation is that the gas flows play a larger role than the voltage gradient on the ion transport in this region, and thus trapping does not occur. The fragmentor voltage defines an acceleration region at the entrance of the high-pressure funnel, and its effect will be described in the section on CIU. For soft conditions, its value is set slightly above the HP funnel entrance absolute value.

Trapping funnel RF amplitude. The high-pressure funnel and trap funnel have also radiofrequencies (1.5 MHz for the high-pressure funnel, 1.2 MHz for the trapping funnel). May et al. described that both RF amplitudes must be decreased to 80 V p-p to have softer conditions. We reproduced this trend when operating the drift tube in nitrogen (Figure S2C), and chose 100 V p-p to optimize the signal. However, in helium, the situation is different: from 80 V p-p to 160 V p-p , no significant ion activation was observed.

Unfolding is only noticed at 180 V p-p and higher (in nitrogen, unfolding is noticeable already at 140 V p-p ).

The reason for the difference is that when helium is used in the IM tube, helium is also injected in the funnels to avoid nitrogen contamination in the tube, and ions move faster overall. Thus, in helium, similar RF amplitudes have a lower effect on ion activation than in nitrogen.

It is surprising that the trap funnel RF amplitude has a greater effect on ion activation in nitrogen than in helium, while the DC gradients in the same region have a greater effect in helium than in nitrogen. A possible origin of the difference could be a different positioning of the ion cloud in the trapping region, depending on the ion gas, for given RF amplitudes. If radial ion placement differs according to the gas composition, it is possible that a given DC voltage different has different effects for the ions in terms of actual electric field. However, to test whether this hypothesis explains the observations, simulations of ion trajectories in presence of collisions with the background gases would be required.

We will describe below that it is possible to carry out CIU experiments in the trapping region. To do that, however, the trap funnel RF amplitude has to be high enough, presumably to confine the ions in the region where the DC gradient is applied. Also, for larger ions the RF amplitudes have to be increased (for example, 200 V p-p is optimum for native bovine serum albumin (66 kDa) transmission, data not shown).

The final choice of the RF amplitudes will depend on the type of experiment: 100 V p-p for native MS on fragile molecules in nitrogen, and 160 V p-p or higher in helium and for CIU. We also found that the HP funnel RF amplitude had little effect on transmission and softness (Figure S3): 100 V p-p and 180 V p-p give the same results. Again, gas flows may dictate ion transport more than voltages in the high-pressure funnel.

Trapping and release times. We noticed an effect of the trap release time (the time during which the trap voltages are set to the green ramp in Figure 1, to push the ions towards the IM) on the arrival time distribution. Short trap release times are supposed to help bunch the ion packet towards the IM with a short time spread (contributing minimally to the width of t 0 ). However, with trap release times shorter than 200 µs, ubiquitin gets more activated and the total ion signal decreases (Figure 2E). A possible explanation is that the fraction of the ion population that had the opportunity to exit the trap during short release times had to do so quickly, and thus traveled faster and got activated more. Longer times than 200 µs did not change the allure of the peaks. The same effect was observed in nitrogen, and the optimum for softness was 250 µs. Again, the difference comes from the slower movement of ions in nitrogen. The trap fill time (Figure 2F) did not influence the softness. However, the ion signal decreases at long trap fill times. Normally the ion signal should increase linearly with the fill time unless there is space charge, as described on a similar trap with one entrance grid and two exit grids. [START_REF] Clowers | Enhanced Ion Utilization Efficiency Using an Electrodynamic Ion Funnel Trap as an Injection Mechanism for Ion Mobility Spectrometry[END_REF] The same phenomenon was observed on a solution ten times less concentrated (0.075 µM, see Figure S4), giving ten times lower signal with the standard ESI source, so the phenomenon is not due to space charge. The origin of this phenomenon is unknown.

Influence of the electrospray source parameters

We also examined how the electrospray source parameters influence the activation of ubiquitin 7+ . Here the effects of the electrospray process (at stake in the intermediate phase consisting of the dense solvent plume) and of ion activation (desolvated ions undergoing collisions) may be combined. The nebulizer gas flow had no effect on the apparent activation. It had only an effect on the ion signal and the optimum was at 9 psig. This parameter should be optimized based on ion signal for each solvent.

In contrast, the drying gas flow had an influence on the apparent ion activation. In the standard electrospray source, a heated drying gas is coming from the entrance capillary, countercurrent to the spray. At 220 °C, high flows cause substantial activation, and low flows resulted in lower ion signals. The optimum drying gas flow, for ubiquitin infused at 190 µL/hour, was 1.5 L/min.

The drying gas temperature, when decreased, also resulted in decreased ion signals, without necessarily decreasing the apparent ion activation. The droplets interact only very transiently with the heated nebulizer gas and undergo endothermic evaporation. Consequently, the effect of gas heating is not equivalent to heating the solution, and gas temperatures higher than the solution melting temperature can be used. However, too low temperatures were very detrimental to the IM signals: over a time scale of several minutes, the IM peaks started to shift to longer drift times, and even when going back to the usual temperature of 220 °C, the shift persisted for over an hour. Our interpretation is that when the drying gas temperature is too low, significant amounts of electrospray solvent enters the funnels and eventually contaminate the drift tube, where even trace amounts of polar molecules in the helium bath gas slow down the ions. Given that the drying gas temperature did not affect the apparent harshness of the conditions, we recommend using sufficiently high temperatures in electrospray, to avoid solvent contamination in the pressure differential. We found that solvent-dependent contamination problems in the tube are more likely to occur when the drift tube is operated in helium, compared to nitrogen.

Finally, we compared three different ion sources: the standard ESI source (results above), the JetStream™ source (Agilent Technologies), and static nanospray ionization using extra coated L borosilicate capillaries (ThermoFischer Scientific, Illkirch, France). Figure S5 shows the comparison for Ubiquitin 7+ , with nitrogen as drift gas. The standard ESI source appears slightly softer. Similar conclusions were reached by Konermann's group on a Waters Synapt ion mobility mass spectrometer. [START_REF] Sun | Protein Structural Studies by Traveling Wave Ion Mobility Spectrometry: A Critical Look at Electrospray Sources and Calibration Issues[END_REF]

Comparison of ubiquitin sprayed from different solvents

To compare ubiquitin sprayed from different solvents, we thus used the drift tube in nitrogen. After starting the injection of each solvent mixture for at least five minutes, we controlled the pressure differential with the flow controller off then on again, and then recorded the data presented here. The optimum source and transfer conditions of Table 1 were used for all solvent mixtures. The resulting arrival time distributions are shown in Figure 3.

Figure 3A shows the arrival time distribution obtained in water/methanol and 0.2% formic acid, i.e. the denaturing conditions used by May et al on the Agilent 6560. [START_REF] May | Conformational landscapes of ubiquitin, cytochrome c, and myoglobin: Uniform field ion mobility measurements in helium and nitrogen drift gas[END_REF] Even with the optimized soft instrument tuning, the conformations under this charge state are mostly denatured. In contrast, when using 1% acetic acid instead of 0.2% formic acid (Figure 3B), which are the conditions used by Russell's group on the Waters Synapt, [START_REF] Chen | How Closely Related Are Conformations of Protein Ions Sampled by IM-MS to Native Solution Structures?[END_REF] despite the charge state distribution is also centered on high charge states, the conformations under the 7+ charge states are predominantly compact, which may be due to a small fraction still with a native fold in solution. The apparent discrepancy between the results of May et al. and Russell et al. was thus mainly due to the solvent choice.

In the interest of native MS, we also investigated native supercharging conditions, here 0.5% sulfolane in 150 mM NH 4 OAc (Figure 3C). The 7+ ion is abundant and its arrival time distribution is mainly constituted from a compact conformation, and the conditions appear even softer than in H 2 O/acetic acid (Figure 3F). However, the peak is slightly offset to a higher collision cross section (1337 Ų) compared to all sample preparations without sulfolane (1262 Ų, standard deviation: 9 Ų). In comparison, the compact peak obtained from 50 mM NH 4 OAc (i.e., Russell's native conditions, [START_REF] Chen | How Closely Related Are Conformations of Protein Ions Sampled by IM-MS to Native Solution Structures?[END_REF] Figure 3D) or 20 mM NH 4 OAc (Figure 3E) is similar to the one obtained from aqueous acetic acid (i.e., May's native conditions, [START_REF] May | Conformational landscapes of ubiquitin, cytochrome c, and myoglobin: Uniform field ion mobility measurements in helium and nitrogen drift gas[END_REF] Figure 3F). The supercharging mechanisms in the presence of NH 4 OAc are still a subject of debate, [START_REF] Hogan | Ion mobility-mass spectrometry of phosphorylase B ions generated with supercharging reagents but in charge-reducing buffer[END_REF][START_REF] Sterling | Supercharging protein complexes from aqueous solution disrupts their native conformations[END_REF][START_REF] Chingin | Soft supercharging of biomolecular ions in electrospray ionization mass spectrometry[END_REF][START_REF] Loo | What protein charging (and supercharging) reveal about the mechanism of electrospray ionization[END_REF] and our results suggest that although the conformations are compact, they are not necessarily identical to the native ones. These preliminary results call for further systematic studies, which are beyond the scope of this paper and will be reported elsewhere.

Collision-induced unfolding (CIU) experiments

We found two parameters which could cause substantial activation: the fragmentor voltage and the trap entrance grid delta. The fragmentor is accelerating the ions between the extremity of the coated glass capillary and the entrance of the high pressure funnel. Figure 4A shows the resulting CIU plot: at 450 V the abundance of the intermediate state becomes slightly larger, and at 490 V an extended state appears.

Figure 4B shows that CIU plots can also be obtained by varying the trap entrance grid delta, which is the voltage difference between the trap entrance grid "low" (during trap filling), and "high" (during storage and release, see Figure 1). This means that, after filling, the ions in the trap are subjected, on the entrance side, to a sudden voltage difference equal to (trap entrance grid delta + trap entrance grid low -trap entrance), and that this voltage can activate the entire ubiquitin 7+ population to unfold it. Note that for carrying out CIU experiments using the trap entrance grid delta voltage, the trap funnel RF amplitude has to be increased to 160 V p-p . Too low trap RF amplitudes result in signal losses at some voltages, and aberrant CIU plots if normalization is done spectrum per spectrum (see Figure S6).

Comparing Figures 4A and 4B also reveals that the activation processes are not equivalent in the two regions. When ubiquitin 7+ is activated in the trap, three distinct extended conformations are produced, eventually two at the highest voltages. This is in line with previous reports by IMS-IMS, [START_REF] Koeniger | Transfer of Structural Elements from Compact to Extended States in Unsolvated Ubiquitin[END_REF][START_REF] Shi | Solution dependence of the collisional activation of ubiquitin [M + 7H](7+) ions[END_REF] CIU in a Waters Synapt, [START_REF] Wagner | Increasing Ubiquitin Ion Resistance to Unfolding in the Gas Phase Using Chloride Adduction: Preserving More "Native-Like" Conformations Despite Collisional Activation[END_REF] and by activated TIMS. [START_REF] Ridgeway | Microheterogeneity within conformational states of ubiquitin revealed by high resolution trapped ion mobility spectrometry[END_REF] When activated in the fragmentor region, however, a single extended population is observed, which is broader and at a median arrival time compared to the conformers found by activation in the trap. We conjecture that residual solvent vapor may still be present in the high pressure funnel where the fragmentor operates, and that ion-molecule reactions could contribute rearranging the extended conformers. This hypothesis would warrant further investigation, depending on source conditions and on the solvent, and if confirmed, comparing CIU in the fragmentor and in the trap could be a useful method to compare CIU in partially solvated vs. gas phase environments.

Optimization of the post-IM region for fragile non-covalent complexes

When fragmentation of very fragile complexes must be reduced also behind the drift tube, the voltage gradients from the rear funnel down to the TOF region can be lowered. After choosing the softest parameters before the IM, we optimized the post-IM region using the ammonium-bound bimolecular Gquadruplex [(dGGGGTTTTGGGG) 2 •(NH 4 + ) 3 -8H] 5-, which served to benchmark several instruments, [START_REF] Balthasart | Ammonium ion binding to DNA G-quadruplexes: do electrospray mass spectra faithfully reflect the solution-phase species?[END_REF] and the results are shown in Figure 5. In addition to the default parameters provided by the manufacturer following installation, we devised two sets of voltage gradients: one is the softest that is still compatible with IMS for our complexes, while minimizing fragmentation ("very fragile" tuning parameters), and one that is a compromise giving higher transmission and better IM resolution. The voltage gradients are shown in Figure 5A. Again, some of the applied voltages are differences between an entrance and a delta voltage entered in the data acquisition software. By lowering the entire voltage gradient, we could obtain the intact three-ammonium complex as the major species when the drift tube is in helium (Fig. 5D), and even the five-ammonium complex, which is also specific, [START_REF] Balthasart | Ammonium ion binding to DNA G-quadruplexes: do electrospray mass spectra faithfully reflect the solution-phase species?[END_REF] with the drift tube in nitrogen (Fig. 5G). The choice of the voltage set depends on the application. If post-IM fragmentation is to be avoided at all costs, the shallowest possible gradient is recommended, but this comes at the expense of ion signal and IM resolution, as shown in Figure S7 on ubiquitin 7+ . With too shallow gradients, the ions wander around longer, so the time spent outside the IM drift tube (t 0 ) increases, and diffusion increases. With extremely shallow gradients, one may even reach a limit where the ions are trapped in hexapoles, and all IM resolution is lost (MS analysis is still feasible). For studying purely intramolecular conformations (provided that no multimer dissociation is interfering), the default steeper gradients should be used, to maintain the best possible IM resolution.

Conclusions

Optimizing the Agilent IMS-Q-TOF for native MS, especially when the drift tube is operated in helium, required departing significantly from the recommended default settings. After examining systematically the combined effects of nearly forty parameters, we obtained conditions allowing to obtain mainly the native ubiquitin 7+ conformation, and the intact fragile G-quadruplex [(dGGGGTTTTGGGG) 2 •(NH 4 + ) 3 -8H] 5-. The main difference between our IM profiles and those reported by May et al. [START_REF] May | Conformational landscapes of ubiquitin, cytochrome c, and myoglobin: Uniform field ion mobility measurements in helium and nitrogen drift gas[END_REF] come from (1) the solvent used and (2) the entrance grid potential upon ion release. The voltage difference between the trap entrance grid and the trap entrance is a key parameter to tune ion activation in the trapping region.

Some tuning parameters differ in helium and in nitrogen because the ions move faster in helium, and slightly softer conditions could be obtained in nitrogen. We also demonstrated that collision-induced unfolding (CIU) experiments could be conducted in two different ways: by increasing the fragmentor voltage or the trap entrance grid delta voltage. Both types of CIU data are interesting, given that the fragmentor operates in a region where solvent vapors may still be present, whereas the trapping region is closer to pure gas-phase conditions. Understanding all these parameters will be useful for several kinds of native MS applications, for example to choose conditions that minimally perturb the structures coming from the solution, to activate them intentionally, or to minimize fragmentation both before and after the IM in order to correctly assign the mobility peaks. In future work, the optimized tuning described herein can be exploited to investigate particularly fragile complexes, for example maintained by only a few weak non-covalent interactions, or with high charge densities and thus higher Coulomb repulsion between subunits. 2) on the arrival time distribution of ubiquitin 7+ (produced from 99% H 2 O, 1% acetic acid, drift tube in 3.89 Torr helium with V = 390 V). The measured collision cross section is unchanged, but the t 0 value changes (5.21 ms for the softest conditions, 4.83 ms for the compromise tuning, 3.70 ms for the default installation tuning).

Figure 1 :

 1 Figure 1: DC voltage profile before the ion mobility (IM) drift tube entrance, with parameters optimized for 3.89 Torr helium in the drift tube and a pressure differential of 0.26 Torr compared to the trapping funnel. The parameters are listed in Table1.

Figure 2 :

 2 Figure 2: Influence of various tuning parameters on the arrival time distribution of Ubiquitin 7+ , sprayed from a 0.75 µM solution in 99% H 2 O and 1% acetic acid, with the drift tube operated in 3.89 Torr helium.The same number of scans were summed in all cases. The drift tube was operated with V = 390 V. The peak at ~25 ms corresponds to the native form (CCS He = 981 Ų; standard deviation was 7 Ų from 10 independent measurements). The optimum value is in bold. Effects of A) voltages on the whole trapping region (see inset of Figure1), the values written being the trap entrance grid low-trap entrance-trap exit-trap exit grid 1 low-trap exit grid 2 low; B) trap funnel delta; C) HP funnel RF and front funnel RF, both set at the same value; D) trap release time; E) trap fill time; F) HP funnel delta and fragmentor voltage; G) electrospray nebulizer pressure; H) dry gas flow; I) dry gas temperature effect, in order of recording. Note that after lowering the temperatures for a while, the system takes a very long time to recover its initial condition.

Figure 3 :

 3 Figure 3: Arrival time distribution of the ubiquitin 7+ and charge state distributions obtained from different solvents (drift tube in 3.89 Torr nitrogen, V = 1390 V): A) H 2 O/MeOH/formic acid (49.9/49.9/0.2 % vol); B) H 2 O/MeOH/acetic acid (49.5/49.5/1 % vol); C) 150 mM NH 4 OAc, 0.5% sulfolane; D) 50 mM NH 4 OAc; E) 20 mM NH 4 OAc; F) H 2 O/acetic acid (99/1 % vol).

Figure 4 :

 4 Figure 4: Collision-induced unfolding (CIU) of ubiqutin 7+ , with the drift tube in 3.89 Torr nitrogen (optimized voltages from Table 1, V = 1390 V), obtained by changing (A) the fragmentor voltage (trap funnel RF amplitude = 100 V p-p ), or (B) the trap entrance grid delta voltage (trap funnel RF amplitude = 160 V p-p ).

Figure 5 :

 5 Figure 5: (A) Illustration of the DC voltage gradient behind the drift tube in negative mode, with the three sets of voltages listed in Table 2. (B-G) ammonium adduct distribution on the bimolecular Gquadruplex [(dG 4 T 4 G 4 ) 2 •(NH 4 + ) n -(5+n)H + ] 5-, obtained in helium (B-D) with the three different gradients (B: default, C: compromise, D: very fragile) and in nitrogen (E-G, same color code).

Figure S1 :

 S1 Figure S1: Comparison of the pre-IM DC voltage gradient in the default tuning parameters and in the tuning parameters optimized for soft conditions when the drift tube is operated in 3.89 Torr helium and the trapping funnel pressure is 3.73 Torr.

Figure S2 :

 S2 Figure S2: Influence of tuning parameters on the arrival time distribution of Ubiquitin 7+ , sprayed from a 0.75 µM solution in 99% H2O and 1% acetic acid, with the drift tube operated in 3.89 Torr nitrogen. The drift tube was operated with V = 1390 V. The peak at ~25 ms corresponds to the native form (CCS N2 = 1262 Ų; standard deviation was 9 Ų from 5 independent measurements in the optimum conditions, which are indicated in bold). Effects of A) voltages on the whole trapping region (see inset of Figure 1), the values written being the trap entrance grid low-trap entrance-trap exit-trap exit grid 1 low-trap exit grid 2 low; B) trap funnel delta; C) HP funnel RF and front funnel RF, both set at the same value; D) trap release time.

Figure S3 :

 S3 FigureS3: Influence of the high pressure funnel RF amplitude and trap funnel RF amplitude on the arrival time distribution of ubiquitin 7+ ions, sprayed from a 0.75 µM solution in 99% H2O and 1% acetic acid, with the drift tube in nitrogen. Only the trapping funnel RF amplitude is responsible for the ion activation.

Figure S4 :

 S4 Figure S4: Influence of the trap fill time on the arrival time distribution of ubiquitin 7+ ions, sprayed from (A) a 0.75 µM or (B) a 0.075 µM solution in 99% H2O and 1% acetic acid, with the drift tube in helium. The same number of scans were summed in each case.

Figure S5 :

 S5 Figure S5: Arrival time distribution of ubiquitin7+ , sprayed from a 0.75 µM solution in 99% H2O and 1% acetic acid, with optimization instrumental parameters (see Table1) and the drift tube in 3.89 Torr nitrogen. (A) Standard ESI source, with drift gas at 1.5 L/min and 220 °C, nebulizer gas at 9 psi, spray voltage at 3500 V. (B) JetStream™ source, with with drift gas at 1.5 L/min and 220 °C, nebulizer gas at 9 psi, spray voltage at 3500 V, jet gas flow at 2 L/min and 30°C, and nozzle voltage at 1000 V. (C) Nanospray source with manually cut coated borosilicate capillary, spray voltage at 1100 V, no nebulizer gas, and drying gas at 13 L/min and 30 °C (similar to May et al.[START_REF] Schalley | Supramolecular chemistry goes gas phase: the mass spectrometric examination of non-covalent interactions in host-guest chemistry and molecular recognition[END_REF] ). Note that the nanospray cone diverts most of the drift gas flow.

  Figure S5: Arrival time distribution of ubiquitin7+ , sprayed from a 0.75 µM solution in 99% H2O and 1% acetic acid, with optimization instrumental parameters (see Table1) and the drift tube in 3.89 Torr nitrogen. (A) Standard ESI source, with drift gas at 1.5 L/min and 220 °C, nebulizer gas at 9 psi, spray voltage at 3500 V. (B) JetStream™ source, with with drift gas at 1.5 L/min and 220 °C, nebulizer gas at 9 psi, spray voltage at 3500 V, jet gas flow at 2 L/min and 30°C, and nozzle voltage at 1000 V. (C) Nanospray source with manually cut coated borosilicate capillary, spray voltage at 1100 V, no nebulizer gas, and drying gas at 13 L/min and 30 °C (similar to May et al.[START_REF] Schalley | Supramolecular chemistry goes gas phase: the mass spectrometric examination of non-covalent interactions in host-guest chemistry and molecular recognition[END_REF] ). Note that the nanospray cone diverts most of the drift gas flow.

Figure S6 :

 S6 FigureS6: Influence of the trap funnel RF amplitude on the CIU results obtained when varying the trap entrance grid delta voltage. When normalizing the arrival time distribution at each voltage, the CIU plots look strange at low trap funnel RF (120 V p-p ). In reality, the signal intensity is lost at some specific voltage ranges (see bottom row where the normalization is done over the entire experiment, not spectrum per spectrum). As the RF amplitude is increased, there is no more signal loss, but the activation at low voltage is becoming prominent. For ubiquitin7+, 160 Vp-p presents a good comprmise to carry out CIU experiments.

Figure S7 :

 S7 Figure S7: Effect of changing the voltage gradient behind the IM drift tube (parameters, see Table2) on the arrival time distribution of ubiquitin 7+ (produced from 99% H 2 O, 1% acetic acid, drift tube in 3.89 Torr helium with V = 390 V). The measured collision cross section is unchanged, but the t 0 value changes (5.21 ms for the softest conditions, 4.83 ms for the compromise tuning, 3.70 ms for the default installation tuning).

Table 1 .

 1 Tuning parameters (positive mode) in the pre-IMS zone. All voltages are floating on the ion mobility tube entrance. The parameters are transposable to the negative mode by reversing the signs.

	Parameter

Installation by Agilent May et al. 21 Optimized on native Ubi 7+ , drift tube in He Optimized on native Ubi 7+ , drift tube in N 2

	Source: gas temperature	325 °C	25 °C	220 °C	220 °C
	Source: drying gas	5.0 L/min	13 L/min	1.5 L/min	1.5 L/min
	Source: nebulizer pressure	20 psig	None	9 psig	9 psig
	Source: capillary	4000 V	Nanospray	3500 V	3500 V
	Optics 1: Fragmentor	400 V		300 V	300 V
	IM front funnel: high	150 V		110 V	110 V
	pressure funnel delta
	IM front funnel: high	150 V p-p	80 V p-p	100-180 V p-p	100-180 V p-p
	pressure funnel RF
	IM front funnel: trap funnel	180 V		140 V	160 V
	delta
	IM front funnel: trap funnel	150 V p-p	80 V p-p	160 V p-p	100 V p-p for
	RF				softness, 160
					V p-p for CIU
	IM front funnel: trap funnel	10 V		10 V	10 V
	exit
	IM trap: trap entrance grid	95 V		70 V	90 V
	low
	IM trap : trap entrance grid	10 V		2 V	2 V
	delta
	IM trap: trap entrance	91 V		69 V	89 V
	IM trap: trap exit	90 V		67 V	87 V
	IM trap: trap exit grid 1 low 88 V		64 V	84 V
	IM trap: trap exit grid 1	6 V		5 V	5 V
	delta
	IM trap: trap exit grid 2 low 87 V		63 V	83 V
	IM trap: trap exit grid 2	10 V		9 V	9 V
	delta
	Acquisition: Trap fill time	20 000 µs		1000 µs	1000 µs
	Acquisition: Trap release	150 µs		200 µs	250 µs
	time

Acknowledgements

The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) / ERC grant agreement n° 616551 (project DNAFOLDIMS to VG).