Deciphering of polycationic carbohydrate based non-viral gene delivery agents by ESI-LTQ-Orbitrap using CID/HCD pairwise tandem mass spectrometry

Deciphering of polycationic carbohydrate based non-viral gene delivery agents by ESI-LTQ-Orbitrap using CID/HCD pairwise tandem mass spectrometry† C´ edric Przybylski,‡* a Juan M. Benito, b V´ eronique Bonnet, d Carmen Ortiz Mellet c and Jos´ e M. Garc´ ıa Fern´ andez b For almost three decades, gene therapy has been gaining interest to efficiently treat some severe diseases. In such context, the discovery of an efficient non-viral gene carrier to deliver genetic material into targeted cell nuclei is of prime importance. Numerous synthetic vectors that have been designed exhibit high transfection efficiency but also suffer from extensive cytotoxicity, thus justifying efforts to synthesize more bio-compatible ones, for example, with carbohydrate scaffolds. In this sense, cyclodextrins (CDs) are well known to present low to very low cytotoxicity in humans and have potential, after polycationization, to serve as suitable compaction/transfection agents for RNA/DNA. However, such polycationic CDs must be accurately characterized to establish a straightforward structure–biological activity relationship which is guided by the nitrogen/phosphorus ratio (N/P). In the study herein, we demonstrated that electrospray-(tandem) mass spectrometry (ESI-(MS)MS) combining Collision Induced Dissociation (CID) and Higher Collision induced Dissociation (HCD) is a useful tool for such synthetic agent characterization. The suitability of CID/HCD pairwise combination was investigated for the structural deciphering of five representative members of a polycationic cyclodextrin library. Our approach allows for easy access to content, type and localisation of amino groups thereby offering a useful tool to correlate the synthetic delivery agent with effective compaction of oligo-/polynucleotides. 1. Introduction Nowadays, a number of therapeutic strategies rely on crossing biological barriers to deliver active drugs directly to the target cells. In some cases, such as during gene therapy, the success of the clinical assay is highly dependent of efficient and safe delivery systems. This issue was soon recognized and still remains a challenge for the past three decades. Due to their natural ability to infect cells, viruses (viral capsids), aer oriented modication of their genome, represent attractive vectors for gene delivery. Nonetheless, concerns exist about their immunogenicity and scaled up production. Indeed some of them have been revealed as insecure in advanced clinical trial stages. 1–3 Consequently to this bottleneck, alternative ways were explored taking into account the poor stability of DNA in vivo,to design appropriate vectors aiming both to protect the genetic payload from degradation during transport and to enhance cell permeability. 4 Most of the developed strategies are based on tailoring polycationic agents which self-assemble in the presence of polyanionic nucleic acids to form small (nano-) particles. Polycations have garnered tremendous interest as designed carriers due to their low host immunogenicity and ease of preparation, for example, built from cationic lipid, 5–8 or also presenting higher molecular weight. 8 Unfortunately, highmolecular weight polycations usually exhibit high transfection efficiency, but possess high cytotoxicity (e.g. polyethyleneimine, PEI). Consequently, efforts have been made to obtain biocompatible architectures, which can potentially serve as carriers. Some cationic synthetic polymers used as vectors have emerged as a promising alternative because of their scalable production, as well as synthetic ease of tuning their size, structure, and functionality. 3,9 A number of amine-containing synthetic polymers have been explored to form a polyplex, including for a Universit´ ed’Evry-Val-d’Essonne, Laboratoire Analyse et Mod´ elisation pour la Biologie et l’Environnement, CNRS UMR 8587, Bˆ atiment Maupertuis, Bld F. Mitterrand, F-91025 Evry, France. E-mail: cedric.pr[email protected] b Instituto de Investigaciones Qu´ ımicas (IIQ), CSIC –Universidad de Sevilla, Am´ erico Vespucio 49, Isla de la Cartuja, E-41092 Sevilla, Spain c Departamento de Qu´ ımica Org´ anica, Facultad de Qu´ ımica, Universidad de Sevilla, E- 41012 Sevilla, Spain d Universit´ e de Picardie Jules Verne, Laboratoire de Glycochimie, des Antimicrobiens et des Agroressources, CNRS UMR 7378, 80039 Amiens, France †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra14508f ‡Present address: Universit´ e Pierre et Marie Curie, Institut Parisien de Chimie Mol´ eculaire, CNRS UMR 8232, 4 place Jussieu, 75252 Paris Cedex 05, E-mail: [email protected]r Cite this: RSC Adv.,2016,6, 78803 Received 4th June 2016 Accepted 5th August 2016 DOI: 10.1039/c6ra14508f www.rsc.org/advances This journal is © The Royal Society of Chemistry 2016 RSC Adv.,2016,6,78803–78817 | 78803 RSC Advances PAPER Published on 05 August 2016. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 09/09/2016 14:18:35. View Article Online View Journal | View Issue example, the paradigmatic PEI, 10,11 poly-L-lysine (PLL), 12 poly(2- (dimethylamino)ethyl methacrylate) (PDMAEMA) 13 or also polyamidoamine (PAMAM) dendrimers. 14 Natural polysaccharides can also be used as a scaffold like chitosan, alginate, dextran, pullulan, cellulose derivatives, cycloamylose, or also hyaluronic acid which are particularly appealing in such case because they are sustainable, non-toxic, and biodegradable. 15–20 Unfortunately, polycationic based (bio)polymers are quite polydispersed. 21 Alternatively, molecularly well-dened entities based on macrocyclic scaffolds, 22 such as calixarenes, 23,24 pillarenes, 25 or cyclodextrins (CDs), 26,27 offer unprecedented opportunities. In this regard, CDs which are cyclic carbohydrates, composed of 6 (a), 7 (b) and 8 (g)a-(1 /4) D-glucopyranoside units present several advantages as a well dened structure, high purity, commercial availability as well as low cytotoxicity, and hence are good candidates to serve as a multivalent core for gene delivery agent synthesis. CDs have been elegantly exploited as a molecular scaffold for the design of gene carriers to enhance the gene delivery capabilities of rst generation lipidic or polymeric nonviral vectors. 7,28,29 Such versatility of the molecules with synthetic methodologies permits installing a large array of regioselective functional elements graed on the CD structure as monodisperse polycationic CDs with 28,30–37 or without amphiphilic features. 34,35,38–41 Nevertheless, even considering a well-dened starting scaffold, the synthesis of more complex structures must be controlled with adequate characterization methods. Due to both the nature and number of graed functions, this remains a challenging task. Since the last two decades, the gentle ionization process electrospray coupled to mass spectrometry (ESIMS) has rapidly gained attraction as a suitable technique to probe sample content. ESI-MS exhibits high sensitivity involving both low sample consumption and residue detection, as compared to other usual methods such as NMR. Moreover, MS 2 and beyond allows for performing structural deciphering via sequential fragmentation, making ESI-MS n a major tool for agent characterization. Various carbohydrate species have been largely studied by MS/MS employing various dissociation modes mostly in the low energy regime like collision induced dissociation (CID), which is the most used fragmentation mode for carbohydrates, 42 and infrared multiphoton dissociation (IRMPD). 43 Structural deciphering can also be achieved by electron assisted methods such as electron capture dissociation (ECD), 44 electron transfer dissociation (ETD), 45 electron detachment dissociation (EDD), 46 or also ultraviolet photodissociation (UVPD). 47 Recently, the usefulness of Higher Collision Dissociation (HCD) during ESI-MS 2 experiments was described for the rst time for linear and cyclic carbohydrate characterization, exhibiting complementary information to the usual CID. 48,49 Nevertheless, to our knowledge, no study has reported the use of ESI-MS and ESI-MS/MS analysis for polycationic carbohydrates whatever the dissociation mode. In the study herein, we investigated ve variously synthesized polycationic cyclodextrin based preparations having demonstrated variable ability to form complexes with RNA/DNA and inducing its compaction. 34,35 We are committed to conrm the homogeneity of samples, probe and compare their gas phase stability in both CID and HCD mode as a function of charge states as well as to achieve the most accurate sequencing. The goal is to establish the benets and limitations of such a pairwise combination to serve as a suitable tool to correlate structural determinants to the biological activity relationship. 2. Experimental section 2.1 Chemicals and reagents Methanol (MeOH) used for sample preparation was of HPLC grade and was purchased from VWR (West Chester, PA, USA). Ultrapure water was of ultrapure quality, obtained from a MilliQ apparatus (Millipore, Milford, USA). 2.2 Samples Synthesis of per-6-modied-b-CD. Per-6-amino-b-CD (agent 1), per-6-cysteaminyl-b-CD (agent 2), per-6-thioureidocysteaminyl- b-CD (agent 3), per-6-aminoethylthioureidocysteaminyl-b-CD (agent 4) and per-6-dithioureidocysteaminyl-b-CD (agent 5) (Fig. 1) were realized as previously described. 34,50 Samples were prepared at 1mgmL 1 in water/methanol 1/1 (v/v). 2.3 Mass spectrometry ESI-MS experiments were carried out using a LTQ-Orbitrap XL from Thermo Scientic (San Jose, CA, USA) and operated in positive ionization mode, with a spray voltage at 3.7 kV. A water/ methanol 1/1 (v/v) mixture was continuously infused using a 500 mL syringe at 3 mL min 1 ow. Applied voltages were 31 and 115 V for the ion transfer capillary and the tube lens, respectively. The ion transfer capillary was held at 275 C. Resolution was set to 60 000 (at m/z400) for all studies, and the m/zranges were set to 200–2000 in prole mode and in the normal mass range during full scan experiments. Spectra were analyzed using the acquisition soware XCalibur 2.0.7 (Thermo Scientic, San Jose, CA, USA) without smoothing and background subtraction. During MS/MS scans, collision-induced dissociation (CID) was performed with an activation Qvalue of 0.25 and an activation time of 30 ms, which occurred in the linear ion trap analyzer (LTQ) and detection in the Orbitrap in centroid mode. This mode used helium as gas (at a pressure of 2 bar) by adjusting the resonance excitation AC signal of 0–5 V (zero-to-peak) at the secular frequency of the precursor ion m/zvalue. Higher energy collision dissociation (HCD) experiments were performed with an activation time of 100 ms, according to our previous study for oligosaccharides 49 and occurred in an octopole collision cell aligned to the C-trap and detection in the Orbitrap. This dedicated cell is supplied with an rf voltage (2.6 MHz, 500 V p–p )of which the DC offset can be varied by 250 V and crossed with nitrogen gas at a pressure of 5 10 3 mbar. For all modes, the endcap voltage was controlled by the normalized collision energy (NCE) technology, where collision energy was modied for each experiment and is expressed in % of NCE. The peak-to- peak voltage is scaled as V p–p ¼NCE/30 (a(m/z)+b), where aand bare instrument variable parameters set at 0.002 and 0.4, 78804 |RSC Adv.,2016,6,78803–78817 This journal is © The Royal Society of Chemistry 2016 RSC Advances Paper Published on 05 August 2016. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 09/09/2016 14:18:35. View Article Online respectively. The standard range is 0% to 100% corresponding to 0–5/500 V according to the dissociation mode. During the survival yield experiments, the NCE varied from 0 to 70%, while for other experiments in the CID and HCD modes, the NCE was set to the estimated CE 50 value (Table 1) and the precursor selection window was set to 2.8 Da during MS 2 experiments in both dissociation modes. The automatic gain control (AGC) allowed accumulation of up to 10 6 ions for FTMS scans, 2 10 5 ions for FTMSn scans and 3 10 4 ions for ITMSn scans. Maximum injection time was set to 500 ms for both FTMS and FTMSn scans and 100 ms for ITMSn scans. For all scan modes, 1 mscan was acquired. Fragments resulting from fragmentation steps were annotated according to the nomenclature described by Domon and Costello, 51 and adapted for cyclodextrins. 48 3. Results and discussion 3.1 Full MS spectra Obtaining a simple MS spectrum can, in some case, represent a challenging task, especially for polycationic and polyanionic compounds, while it constitutes the rst and the easier step to gain a portrait of the sample content. Neutral carbohydrates are polyol compounds well known to easily form adducts with metal cations (Na + ,K + ,Li + ,.) or ammonium ion (NH 4+ ). 52–54 Notwithstanding, in the study herein the carbohydrates have been chemically modied and present a strong cationic character involving a clear preference for protons. Moreover, the maximal possible charge states depend on several protonable sites, i.e. the number of primary and secondary amines (NH 2 and NH, respectively) but also sulfur (S). For agents 1, 2, 3, 4 and 5, this theoretical number is 7 (NH 2 : 7), 14 (NH 2 : 7, S: 7), 35 (NH 2 : 7, NH: 14, S: 14), 42 (NH 2 : 7, NH: 21, S: 14) and 56 (NH 2 :7, NH: 28, S: 21), respectively. Nevertheless, it can be anticipated that all these groups cannot simultaneously attach protons, mainly due to coulombic repulsion, involving a dedicated rigidity conformation of molecules, steric hindrance and a particular hydration shell. Full MS spectra reveal that agent charge states were between 1+ and 7+ (Fig. 2). A majority of reports suggest that the maximum charge carried by amino based biomolecules such as denaturated protein until z50 kDa, can be directly correlated to their number of basic residues (Arg, Lys and His). 55 However, a previous study especially on higher molecular weight proteins and polyamidoamine based stardust dendrimers gave evidence that features such as solvent, 56 and above all, coulombic repulsion effects directly affect the effective maximum and average charging. 57–59 For high molecular weight proteins, the maximum number of charges (Z max ) observed is smaller and is thought to be obtained by combining the accessibility of basic amino acid side chains near the surface of ESI droplets, and the size and surface tension of the droplets as described by the Rayleigh limit (Z R ). 59–61 Considering a spherical ion in the gas phase, the charge state distribution was dependent on the coulombic repulsion required to lower the energy barrier of proton transfer. From this, Schwartz et al. argued that, since the volume (and hence the resulting mass) varies as r 3 (with rthe radius of the ion) the number of charges on the ion varies as (molecular weight) 2/3 . This study has demonstrated than even if protein sequence is heterogeneous as compared to studied dendrimers, the globular shape of both involves a similar gasphase behaviour and then an identical extent of charging. 57 Another study based on the Dole’s charged residue mechanism demonstrated that the maximum theoretical charge is Z max theo ¼Z R ¼0.0778 (average molecular mass; M w ) 1/2 for native globular proteins and compact starburst dendrimer ions. 58,59 Some other ions representing a more extended structure such as linear chains of polyethylene glycols have experimental Z max (Z max exp) values considerably larger than Z R (Z max exp/Z R >1) which could also be formed from the charged residue mechanism, but with non-spherical drops held together by the polymer backbone. 59 Taking into account the aforementioned equation 58,59 and the experimental spectra, we obtain the following Z max exp/Z R ratio values: 1.14 (3/2.61), 1.31 (4/3.06), 1.89 (7/3.70), 1.77 (7/ 3.94) and 1.65 (7/4.24) for agent 1, 2, 3, 4 and 5, respectively. It is noteworthy that the relative stability of highly-charged Fig. 1 Structures of the five polycationic carbohydrate based non-viral gene delivery agents (agent 1 to 5) studied herein. This journal is © The Royal Society of Chemistry 2016 RSC Adv.,2016,6,78803–78817 | 78805 Paper RSC Advances Published on 05 August 2016. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 09/09/2016 14:18:35. View Article Online species conferred by the 2-aminoethyl-thioureido segment (present in CDs 3, 4 and 5) nicely correlates with the enhanced gene transfer capabilities of amphiphilic CDs containing this functional group (see for instance 34 ). The two last ones exhibit more complex spectra than for others presumably due to more reactive moieties inducing rearrangement and additional loss in the gas phase. However, thanks to both the high resolution power and mass accuracy of the Orbitrap analyzer, this did not impair the correct ascription of ions and their accurate selection for fragmentation. The rst step is to determine the different stability level in the gas phase of molecules as a function of their respective different charge states. 3.2 Probing gas phase stability A measure of the propensity of a structure to dissociate was obtained by determining the collision energy required to obtain 50% of the precursor decomposition (CE 50 value). Here, these values can serve as a tool to probe the strength i.e. the stability of the protonated structures in the gas phase. Such a benchmark was established for every detected charge state i.e. here the number of attached protons. This criterion was previously successfully applied to compare carbohydrate features such as for example the length, 49,62 the cyclic or linear forms, 49 the degree of methylation/number and kind of cations attached. 48 It is well known that Collision Induced Dissociation (CID) is the widely used fragmentation mode to establish the survival yield curves of several molecules. This CID mode operates as a “slow” regime with 1–20 eV at the low ms scale and with approximately 10–1000 collisions per s. Nevertheless, we have previously demonstrated that High Collision Dissociation (HCD), available on a LTQ-Orbitrap instrument, provides an alternative way of fragmentation. Indeed, HCD uses a “triple quadrupole like” fragmentation mechanism which offers ne tuning to monitor agent decomposition such as oligomaltosides and native or Table 1 Recapitulative table of mass accuracy and characteristic CE 50 values obtained in CID and HCD modes for different studied agents according to their respective charge states a Ions Agents m/z Mass accuracy (ppm) CE 50 (%) Experimental Theoretical CID HCD [M + H] + 1 1128.4899 1128.4895 0.4 23.3 10.0 (22 eV) 2 ND 1548.5131 ——— 3 ND 2262.6890 ——— 4 ND 2563.9846 ——— 5 ND 2976.8654 ——— [M + 2H] 2+ 1 564.7488 564.7487 0.2 16.7 14.5 (14 eV) 2 774.7597 774.7605 0.8 16.7 21.0 (26 eV) 3 1131.8482 1131.8485 0.3 19.3 13.1 (23 eV) 4 1282.4958 1282.4962 0.3 18.4 13.2 (31 eV) 5 1488.9355 1488.9366 0.7 22.4 5.7 (22 eV) [M + 3H] 3+ 1 376.8349 376.8350 0.3 10.6 11.0 (7 eV) 2 516.8426 516.8429 0.6 14.8 19.3 (15 eV) 3 754.9013 754.9016 0.4 17.2 15.1 (17 eV) 4 855.3336 855.3334 0.2 16.2 16.5 (23 eV) 5 992.9602 992.9603 0.1 19.3 10.5 (18 eV) [M + 4H] 4+ 1 ND 282.8782 ——— 2 387.8839 387.8841 0.5 8.2 12.0 (6 eV) 3 566.4280 566.4282 0.4 14.7 15.4 (12 eV) 4 641.7523 641.7520 0.5 14.2 17.3 (17 eV) 5 744.9719 744.9722 0.4 15.5 11.2 (13 eV) [M + 5H] 5+ 1 ND 226.5041 ——— 2 ND 310.5089 ——— 3 453.3440 453.3441 0.2 11.2 13.5 (8 eV) 4 513.6035 513.6032 0.6 9.5 16.2 (12 eV) 5 596.1797 596.1793 0.7 10.3 11.1 (9 eV) [M + 6H] 6+ 1 ND 188.9214 ——— 2 ND 258.9254 ——— 3 377.9579 377.9577 0.5 6.7 4.5 (2 eV) 4 428.1703 428.1706 0.7 8.7 11.7 (7 eV) 5 496.9838 496.9841 0.6 8.5 7.4 (5 eV) [M + 7H] 7+ 1 ND 162.0766 ——— 2 ND 222.0800 ——— 3 324.1047 324.1051 0.9 3.5 ND 4 367.1467 367.1473 0.9 8.3 4.2 (2 eV) 5 426.1301 426.1303 0.5 7.7 1.0 (1 eV) a ND, not detected. 78806 |RSC Adv.,2016,6,78803–78817 This journal is © The Royal Society of Chemistry 2016 RSC Advances Paper Published on 05 August 2016. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 09/09/2016 14:18:35. View Article Online methylated CDs. 48,49 HCD mode operates as a “fast”CID mode with 1–200 eV at a high ms scale at an average lower collision rate with approximately 10–100 collisions per s. The determination of CE 50 values was achieved both in CID and HCD dissociation modes. Here, such values can serve as a tool to observe differences in fragmentation susceptibility between various molecules according to a given charge state as well as to study behaviour of a given molecule under different protonation levels i.e. establish a CE 50 /charge states relationship. The NCE was increased by increments of 0.5% from 0% to 70%. Aer NCE screening, the intensity of the precursor ion was recorded as a function of the NCE. The SY was calculated following the equation SY ¼(I X )/(I 0 ), where I X is the intensity of the precursor ion peak aer excitation with a specic percentage of NCE, and I 0 is the intensity of the precursor ion peak aer excitation with 0% NCE. Experimental points were next tted to plot a sigmoid curve, from which CE 50 values were deduced as in previous studies. 48,49 The determination of CE 50 values during both CID and HCD dissociation experiments was achieved for the [M + nH] n+ with 1 #n#7 for each of the ve studied cationic carbohydrates (Table 1). Thorough examination of the results in Table 1 reveals some interesting differences such as the fact that di- and triply charged ions of agent 1 required almost two fold less HCD energy than other agents. Concerning quadruply charged, agent 2 required 2–3 fold CID or HCD less energy than others, while all values are quite close for 5+. Considering the HCD mode, sextuply charged ions exhibit similar trends compared to quadruply charged ones from agent 4 and 5. The case of sextuply charged ions can be set apart, since only three agents show such charge states with agent 3 being z2.3 fold more sensitive to CID fragmentation than others. Moreover, this agent presented too low abundance to be effectively studied by HCD, while agents 4 and 5 exhibited a highly unstable behaviour, leading rapidly to dissociation. In both HCD and CID mode, a quite poor correlation was observed (main regression coefficient R 2 between 0.2032–0.8659 and 0.00231–0.6807 for CID and HCD, respectively, considering at least three points) between the CE 50 values and the m/zfor all carbohydrate derivatives (ESI Fig. S1†). That contrasts with previous results obtained with polymers, 62,63 oligo-/polysaccharides 48,49,62 or peptides. 64 In this last case, it was observed that dispersion of the experimental points was higher for triply charged than singly or doubly charged ions. This can be attributed to more mobile protons and consequently to various peptides gas phase conformations. 64 From the data, the behaviour of each agent towards both their charge states and dissociation mode used is quite contrasted. Nonetheless, two trends can be drawn: (i) the progressive increase of charge states involved moderate and constant reduction dissociation energy (z2–4% and z7–10 eV in CID and HCD, respectively, except for agent 1 where a loss of z6% and 7–8 eV is obtained in CID and HCD, respectively), (ii) based on CE 50 values, agent 4 or 5 appeared as the most stable according to the investigated charge state ranges and according to the dissociation mode like CID and HCD, respectively. Such phenomenon could be attributed to charge delocalization on the 2-aminoethyl-thiourea segments. On the other hand, a clear linear correlation was obtained aer tting the CE 50 value obtained upon HCD with charge states as evidenced by the regression coefficient all between 0.9958 and 0.9987 (Fig. 3). Fig. 2 Full mass spectra of agent 1 (A) 1, 2 (B), 3 (C), 4 (D) and 5 (E). This journal is © The Royal Society of Chemistry 2016 RSC Adv.,2016,6,78803–78817 | 78807 Paper RSC Advances Published on 05 August 2016. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 09/09/2016 14:18:35. View Article Online Here, such results revealed a higher inuence value of the number of protons attached on the CE 50 rather than considering only the m/zratio. Based on the mobile proton theory, well known for peptides and proteins, the higher the number of proton acceptor sites, the higher the probability to induce cleavage. Indeed, due to this strong effective correlation, we can postulate that protons are statistically distributed along molecules in regards to their proton affinity (PA) sites. Among them, we delineate methylamine (CH 3 NH 2 , PA: 899.0 kJ mol 1 ) for agent 1, ethylamine (CH 3 CH 2 NH 2 , PA: 912.0 kJ mol 1 ) for agents 2 to 5, one dimethylamine ((CH 3 ) 2 NH 2 , PA: 929.5 kJ mol 1 ) for agent 4 and one or two N,N-di-methylthiourea moieties ((CH 3 NH) 2 CS, PA: 926.0 kJ mol 1 ) in agent 3 and 4/5, respectively. An additional moiety can be also taken into account, thioruea ((CH 3 NH) 2 CS, PA: 897.3 kJ mol 1 ) but it did not match very well with our structures, since it requires N,N free amino groups. Careful examination of the data in Fig. 3 showed that molecules present different behaviour according to the range of available charge states. In this sense, the slope and intercept of the curve could be used as tools to tentatively discriminate structures. Hence, according to the aforementioned criteria, especially slope, studied molecules can be divided into three sets. The rst contains agents 1 and 2 with a higher slope of 7.5 eV per CS and 10 eV per CS, respectively, showing that they are highly prone to fragment consequent to the increase in charge states. The second set includes agents 3 and 5 which demonstrated a quite similar behaviour with a slope as well of 5.1 and 5.4 eV per CS, respectively, revealing the lowest inuence of charge states upon fragmentation susceptibility (z2–3 lower than for agents 1/2). Finally, a third set was only composed of agent 4 exhibiting an intermediary regime with a slope equal to 4.2 eV per CS. At a glance, the intercept values did not really make sense from a physico-chemical point of view, appearing only as CE 50 values when a 0 net charge is borne i.e. a neutral molecule. Only between agents 1 and 2 were values almost one-third higher (45.7 versus 29.3 eV, all other agents values are between 30.4 and 39.2 eV). That means that, intrinsically, CE 50 values without any charges are close, demonstrating the inuence of the number of charges on the fragmentation propensity as compared to only the initial molecular conformation. Nonetheless, these values could be used to determine experimental appearance energies (AE exp ), i.e. the minimum energy that must be imparted to the molecule to produce an ion, determined in the NCE framework of a given IT-MS as previously demonstrated by Zins et al. 65 That required the application of a single conversion factor from the linear t of 9.8 0.3 kJ mol 1 % 1 between the % NCE scale and kJ mol 1 (i.e. AE exp ¼9.8 % NCE). Aer conversion, we can postulate that the presence of only an amino group (agent 1, AE exp : 287 kJ mol 1 )offers better conditions to rapidly promote efficient ionisation. Next, dithioureidocystenaminyl (agent 5, AE exp : 298 kJ mol 1 ) or an exhibiting thioureidocystenaminyl arm (agent 3, AE exp : 321 kJ mol 1 ) is a little less easy to ionize. This could be due to a more compact structure consequent to the intra-molecular hydrogen bond emphasized due to the two thioureido moieties for agent 5, and lower PA of the single primary amine for agent 1. The presence of aminoethylthioureidocystenaminyl (agent 4, AE exp : 384 kJ mol 1 ) exhibits only one thiourea moiety, as agent 3, but the presence of a ternary amine could involve higher exibility providing a particular conformation of the graed chains. Finally, the cysteaminylated molecules (agent 2, AE exp : 448 kJ mol 1 ) appeared as the most difficult to ionize. However, our experimental AE values must be treated with particular caution, since those previously reported were determined according to the monocharged ion, and not as a extrapolated neutral molecular state. Taking into account both the most abundant charge states as a function of targeted agents (full MS spectra) and determining the relative gas phase stability of each structure according to their protonation state (CE 50 value), better characterization is required by the sequencing of modication/ graed arms. 3.3 Structural deciphering via combined CID and HCD fragmentation experiments Here, even if all detected charges corresponding to intact agents have been fragmented, we only reported the fragmentation spectra of the most intense precursor for each agent according to their respective charge state. If CID is a standard fragmentation technique easily accessible in present-day instruments, it suffers from the low-mass cutoffbottleneck which does not allow the trapping of fragments below z30% of the precursor m/z. This one-third rule is governed by the choice of the instrumental Qvalue depending on the rf amplitude. An alternative way is the recently introduced HCD fragmentation in a not Qdependent matter. With the CID and HCD fragmentation mode, common losses of NH 3 (17.027 mass units, ,at the precursor charge state and -for lower charge states) and H 2 O(18.011 mass units, >at the precursor charge state and Afor lower charge states) were observed. The fragmentation of dicharged ions of per-6-amino-CD (agent 1) mainly yielded to NH 3 and H 2 O losses (Fig. 4A). Nonetheless, enough energy was dissipated to also allow some glycosidic cleavage along the oligosaccharide backbone, but more efficiently by producing monocharged ions rather than dicharged ones. Indeed, only m/z 484.2155 corresponding to [C 7 /Z 6 + 2H] 2+ ion was clearly detected while m/z967.4212, m/z806.3522, m/z645.2828, Fig. 3 Variation of CE 50 HCD values as a function of charge states from the five studied agents. 78808 |RSC Adv.,2016,6,78803–78817 This journal is © The Royal Society of Chemistry 2016 RSC Advances Paper Published on 05 August 2016. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 09/09/2016 14:18:35. View Article Online 484.2155, m/z323.1457 and m/z162.0764 were ascribed to [C 7 / Z n +H] + with 1 < n< 6, respectively. Three additional ion series were also observed, the rst two were attributed to the glycosidic cleavage at m/z341.1566 ([C 7 /Y 2 +H] + ) and m/z180.0871 ([C 7 / Y 1 +H] + )i.e. [GlcNH 2 +H] + or with Y series at m/z323.14576 ([C 7 / Z 2 +H] + ) and m/z162.0764 ([C 7 /Z 1 +H] + )i.e. ([GlcNH 2 H 2 O+ H] + ), and the third one including the loss of one CHNH 2 moiety at m/z884.3626 ([C 7 /Z 6 3H 2 OCHNH 2 +H] + ) and m/z 532.2237 ([M 2H 2 OCHNH 2 + 2H] 2+ ). In HCD mode, similar ions were detected, but exhibited a more balanced spectrum over the whole spectrum. In particular, higher low mass intensity signals corresponding to one building block, and the presence of additional water loss from m/z532.2242 ([M 2H 2 O CHNH 2 + 2H] 2+ ) were observed (Fig. 4B, Bat the precursor charge state). Concerning per-6-cysteaminylated CD (agent 2), in CID, both ammonia and water losses were still highly abundant, associated or not with glycosidic cleavages (Fig. 5A). Nevertheless, these fragments were less abundant as triply charged for example at m/z431.4728, ([C 7 /Z 6 H 2 ONH 3 + 3H] 3+ ) than for a simple charge reduction or as doubly charged at m/z664.2121 ([C 7 /Z 6 + 2H] 2+ ), m/z553.6877 ([C 7 /Z 5 + 2H] 2+ ), m/z434.6418 ([C 7 / Fig. 4 MS/MS spectra obtained for fragmentation of [M + 2H] 2+ of agent 1 in CID (A) and HCD (B). NH 3 loss at the precursor (,) and lower (-) charge states; H 2 O loss at the precursor (>) and lower (A) charge states; additional H 2 O loss (^) from [M 2H 2 OCHNH 2 + 2H] 2+ loss. This journal is © The Royal Society of Chemistry 2016 RSC Adv.,2016,6,78803–78817 | 78809 Paper RSC Advances Published on 05 August 2016. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 09/09/2016 14:18:35. View Article Online Z 4 NH 3 + 2H] 2+ ) and m/z324.1003 ([C 7 /Z 3 NH 3 + 2H] 2+ )as well as a monocharged one like at m/z851.2429 ([C 7 /Z 4 2NH 3 + H] + ), 649.1913 ([C 7 /Z 3 NH 3 +H] + ) and for m/z426.1241 ([C 7 /Z 2 NH 3 +H] + ). It must be noted that this last ion is the rst part of an isotopic cluster (data not shown) including also a dicharged species [C 7 /Z 4 2NH 3 + 2H] 2+ . The presence of an thioether group on the graed arms induces site driven fragmentation which can occur at the aposition of the two sides of the S atom, leading to (CH 2 ) 2 NH (e.g. m/z744.7255 for [M  (CH 2 ) 2 NH NH 3 + 2H] 2+ ) or SH(CH 2 ) 2 NH 2 losses. Nonetheless, in this last case, such loss involves obligatorily the concomitant loss of one water molecule and intra-cyclic cleavage (i.e. m/z 697.2292 and m/z465.1562 for [ 0,2 A 7 /Z 7 SH(CH 2 ) 2 NH 2 H 2 O+ 2H] 2+ and [ 0,2 A 7 /Z 7 SH(CH 2 ) 2 NH 2 H 2 O + 3H] 3+ , respectively). Monocharged monosaccharide with no reducing end exhibiting unsaturation (i.e. coming from sequential rupture of CD’s glycosidic bond) and carrying a cysteaminyl moiety was detected as an intact form at m/z222.0798 ([C 7 /Z 1 +H] + i.e. [GlcS(CH 2 ) 2 NH 2 H 2 O+H] + ) and aer deamination at m/z 205.0528 ([C 7 /Z 1 NH 3 +H] + i.e. [GlcS(CH 2 ) 2 NH 2 H 2 O NH 3 +H] + ) or further dehydration (m/z205.0528, m/z186.0581, m/z168.0478, for additional 1, 2 and 3 water losses, Fig. 5 MS/MS spectra obtained for fragmentation of [M + 3H] 3+ of agent 2 in CID (A) and HCD (B). NH 3 loss at the precursor (,) and lower (-) charge states; H 2 O loss at the precursor (>) and lower (A) charge states. 78810 |RSC Adv.,2016,6,78803–78817 This journal is © The Royal Society of Chemistry 2016 RSC Advances Paper Published on 05 August 2016. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 09/09/2016 14:18:35. View Article Online respectively). Here, the precursor under 3+ upon HCD fragmentation did not signicantly improve the sequence covering of the cyclic carbohydrate and did not produce neither more nor different fragments as compared to CID (Fig. 5B). The CID based MS/MS spectrum of the quadruply charged ion from agent 3 (Fig. 6A) showed few similar ions as that of agent 2, with only the loss of (CH 2 ) 2 NH (e.g. m/z734.8793 and for m/z551.4116 for [M (CH 2 ) 2 NH NH 3 + 3H] 3+ and for [M  (CH 2 ) 2 NH NH 3 + 4H] 4+ , respectively). Otherwise, the presence of the thioureidocystenaminyl moiety induces some particular site driven fragmentation corresponding to the loss of CS(CH 2 ) 2 (NH) 2 (e.g. m/z700.8713 and for m/z540.9213 for [M  Fig. 6 MS/MS spectra obtained for fragmentation of [M + 4H] 4+ of agent 3 in CID (A) and HCD (B). NH 3 loss at the precursor (,) and lower (-) charge states; H 2 O loss at the precursor (>) and lower (A) charge states. This journal is © The Royal Society of Chemistry 2016 RSC Adv.,2016,6, 78803–78817 | 78811 Paper RSC Advances Published on 05 August 2016. Downloaded by Centro de Investigaciones Cientificas Isla de la Cartuja (CICIC) on 09/09/2016 14:18:35. View Article Online