Charge State Distribution Research Articles

Is Native Mass Spectrometry in Ammonium Acetate Really Native? Protein Stability Differences in Biochemically Relevant Salt Solutions.

Ammonium acetate is widely used in native mass spectrometry to provide adequate ionic strength without adducting to protein ions, but different ions can preferentially stabilize or destabilize the native form of proteins in solution. The stability of bovine serum albumin (BSA) was investigated in 50 mM solutions of a variety of salts using electrospray emitters with submicron tips to desalt protein ions. The charge-state distribution of BSA is narrow (+14 to +18) in ammonium acetate (AmmAc), whereas it is much broader (+13 to +42) in solutions containing sodium acetate (NaAc), ammonium chloride (AmmCl), potassium chloride (KCl), and sodium chloride (NaCl). The average charge state and percent of unfolded protein increase in these respective solutions, indicating greater extents of protein destabilization and conformational changes. In contrast, no high charge states of either bovine carbonic anhydrase II or IgG1 were formed in AmmAc or NaCl despite their similar melting temperatures to BSA, indicating that the presence of unfolded BSA in some of these solutions is not an artifact of the electrospray ionization process. The charge states formed from the nonvolatile salt solutions do not change significantly for up to 7 min of electrospray, but higher charging occurs after 10 min, consistent with solution acidification. Formation of unfolded BSA in NaAc but not in AmmAc indicates that the cation identity, not acidification, is responsible for structural differences in these two solutions. Temperature-dependent measurements show both increased charging and aggregation at lower temperatures in NaCl:Tris than in AmmAc, consistent with lower protein stability in the former solution. These results are consistent with the order of these ions in the Hofmeister series and indicate that data on protein stability in AmmAc may not be representative of solutions containing nonvolatile salts that are directly relevant to biology.

Charging of DNA Complexes in Positive-Mode Native Electrospray Ionization Mass Spectrometry.

Native mass spectrometry (nMS) provides insights into the structures and dynamics of biomacromolecules in their native-like states by preserving noncovalent interactions through "soft" electrospray ionization (ESI). For native proteins, the number of charges that are acquired scales with the surface area and mass. Here, we explore the effect of highly negatively charged DNA on the ESI charge of protein complexes and find a reduction of the mass-to-charge ratio as well as a greater variation. The charge state distributions of pure DNA assemblies show a lower mass-to-charge ratio than proteins due to their greater density in the gas phase, whereas the charge of protein-DNA complexes can additionally be influenced by the distribution of the ESI charges, ion pairing events, and collapse of the DNA components. Our findings suggest that structural features of protein-DNA complexes can result in lower charge states than expected for proteins.

The use of second foil stripping in tandem accelerators

The 10 MV FN Tandem at the University of Notre Dame’s Nuclear Science Laboratory has the option for a second foil stripper halfway down its high energy column. With its utilization, users are able to produce beams with higher energies and/or transmission than single foil stripping alone would be capable of achieving. A discussion of the Schiwietz–Grande, Nikolaev–Dmitriev, and Baudinet-Robinet semi-empirical models used to determine the resulting charge state abundances, as well as how they compare to measured charge state distributions is presented. The advantages of a second foil stripper are discussed alongside measurements of the charge state abundances produced. The potential for more interfering beam species of similar magnetic rigidity is also discussed. It was found that for most of the beams tested, second foil stripping allowed higher energies with higher yields than the single terminal foil stripping alone could achieve which can enhance the capabilities of other laboratories using similar accelerator systems.

Spectroscopic analysis of tungsten spectra in extreme-ultraviolet range of 10-480 Å observed from EAST tokamak with full tungsten divertor

Tungsten spectra in extreme ultraviolet (EUV) wavelength range of 10-480 Å have been observed from high-temperature plasmas in Experimental Advanced Superconducting Tokamak (EAST) with full tungsten divertor using four fast-time-response EUV spectrometers of EUV_Short (5-45 Å), EUV_Long_a (40–180 Å), EUV_Long_c (130–330 Å) and EUV_Long_b (270–480 Å) and two space-resolved EUV spectrometers of EUV_Short2_d (45–70 Å) and EUV_Long2_d (40–130 Å). The wavelength of measured spectra is accurately calibrated based on several well-known spectral lines emitted from low-Z (He, Li, C, N and O), medium-Z (Fe and Cu) and high-Z (Mo) impurity ions. Measurements of the tungsten spectra were taken from discharges accompanied with a transient tungsten burst event, which creates a pulsed influx of tungsten atoms into the EAST plasma. The tungsten spectra observed before and after the burst event are carefully analyzed with temporal behavior and radial profile distribution of the tungsten line intensity. As a result, 213 tungsten lines are successfully confirmed in the spectra observed after the tungsten burst, and the results are summarized in tables. These tungsten lines include line identifications of 78 lines of W XXIII - W XLVI (W22+ - W45+) at 10–140 Å and 88 lines of W V - W IX (W4+ - W8+) at 160–480 Å, while 47 tungsten lines at 50–380 Å could not be clarified the transition. In addition, quasi-continuum spectra called unresolved transition array (UTA) from tungsten ions in low- and high-ionization stages are also analyzed in three wavelength ranges of 18–38 Å, 45–70 Å and 150–280 Å at which W XXIII - W XXXVIII (W22+ - W37+), W XXVII - W XLVI (W26+ - W45+) and W VI—W IX (W5+ - W8+) are dominantly emitted, respectively. Through the analysis it is found that charge state distributions of tungsten UTA at 140–220 Å significantly vary with decrease in the electron temperature. Ionization stages of all observed tungsten lines including both isolated and quasi-continuum lines are experimentally reconfirmed by comparing the radial intensity profile with the electron temperature profile. Finally, spectral lines useful for tungsten diagnostic in fusion plasmas are selected and marked in the tables.

Electron-impact double ionization cross sections and rate coefficients for Be-like ions: B+ to Ne6+

We report a detailed analysis of the electron-impact double ionization process for Be-like ions, spanning charge states from B+ to Ne6+. We considered both direct and indirect double ionization processes. For direct double ionization, we calculated the cross sections by considering three different processes: ionization-ionization (II), excitation-ionization-ionization (EII), and ionization-excitation-ionization (IEI). Our results show excellent agreement between the measured cross sections for Be-like-B and Be-like-O ions. The agreement is slightly weaker for Be-like-Ne ions when we take the potential of ionized ions in the distorted-wave method. For other ions for which experimental data are not available, our calculations should provide the missing data. We have summarized the trends of electron-impact double ionization cross sections with atomic number for isoelectronic sequences. Finally, we have provided the Maxwellian and non-Maxwellian rate coefficients for these Be-like ions. The data obtained should be useful for both equilibrium and non-equilibrium charge-state distributions in astrophysical research.

Top-Down Structural Characterization of Native Ubiquitin Combining Solution-Stable Isotope Labeling, Trapped Ion Mobility Spectrometry, and Tandem Electron Capture Dissociation Mass Spectrometry.

Solution-phase hydrogen/deuterium exchange (HDX) coupled to native ion mobility spectrometry mass spectrometry (IMS-MS) can provide complementary structural information about the conformational dynamics of biological molecules. In the present work, the solution-stable isotope labeling (SIL) combined with trapped ion mobility spectrometry (TIMS) in tandem with top-down electron capture dissociation (ECD) is illustrated for the structural characterization of the solution native states of ubiquitin. Four different ubiquitin electrospray solution conditions: (i) single-tip nondeuterated, (ii) theta tip for online SIL HDX, (iii) single-tip SIL-deuterated, and (iv) theta tip for online SIL H/D back exchange (HDbX), were investigated to assess the H/D exchange reactivities of native ubiquitin. The combination of TIMS and ECD in a q-ToF MS instrument allowed for additional inspection of gas-phase HDbX added by top-down fragmentation, revealing the exposed and protected residues with limited scrambling effects (e.g., intramolecular H/D migration). A native charge state distribution (5+ to 7+) and TIMS profiles were observed under the single-tip nondeuterated solution conditions. Mass shift distributions of ∼40, ∼104, and ∼87D were observed when incorporating deuterium for online SIL HDX, SIL HDX, and online SIL HDbX, respectively, while retaining similar conformational states. ECD fragmentation allowed for the localization of the deuterated labeled residues of the peptide fragments, with a sequence coverage of ∼90%, for each of the ubiquitin solution condition. Changes in the TIMS trapping time settings (∼70 to ∼795 ms) were used to determine the H/D back exchange dynamics of native ubiquitin. HDbX-TIMS-q-ECD-MS/MS exhibited H/D back exchanges in the six-residue C-terminal tail as well as around Lys6, Lys11, Lys33, Lys48, and Lys63 residues, indicating that these regions are the most exposed area (less protected hydrogens) of ubiquitin as compared to the rest of the core residues that adopt a compact β-grasp fold (protected hydrogens), which was consistent with the accessible surface area of ubiquitin. The present data highlight for the first time consistency between the solution HDX and gas-phase HDbX-TIMS data for native studies.

CPred: Charge State Prediction for Modified and Unmodified Peptides in Electrospray Ionization.

The mass-to-charge ratio serves as a critical parameter in peptide identification via mass spectrometry, enabling the precise determination of peptide masses and facilitating their differentiation based on unique charge characteristics, especially when peptides are ionized by tools like electrospray ionization, which produces multiply charged ions. We developed a neural network called CPred, which can accurately predict the charge state distribution from +1 to +7 for the modified and unmodified peptides. CPred was trained on the large-scale synthetic training data, consisting of tryptic and non-tryptic peptides, and various fragmentation methods. The model was further evaluated on independent, external test data sets. Results were evaluated through the Pearson correlation coefficient and showed high correlations of up to 0.9997117 between the predicted and acquired charge state distributions. The effect of specifying modifications in the neural network and feature importance was further investigated, revealing the value of modifications and vital peptide properties in holding on to protons. CPreds' accurate predictions of the charge state distribution can play an essential role in boosting confidence in peptide identifications during rescoring as a novel feature.

Effects of anions on the electrospray ionization of proteins in strong acids.

The effect of anions on the positive electrospray ionization (ESI) of proteins in different strong acids with varying pH values from 3 to 1 is studied using high-pressure ESI. Reducing the pH from ∼2 to 1 caused a drastic shift in charge state from a high-charge-state distribution (HCSD) to a narrow low-charge-state distribution (LCSD). The shift in charge state was consistent with the circular dichroism result that showed a conformational change due to the "acid-induced folding" of proteins from an unfolding state to a compact molten globule state. Acids of different anions produced noticeable differences in the average charge for HCSD and LCSD. For HCSD, the average charge was lower than the value typically observed using formic and acetic acids. As for LCSD, the average charge was lower than the "native" charge. The high abundance of acid anion that induces the protein compaction was believed to play a role in charge reduction. The effectiveness of anions to "refold" a highly unfolded protein to a compact state and the propensity to reduce the charge of HCSD for proteins appeared to follow the selectivity series of anions towards the stationary phase in ion chromatography. However, the propensity of anions to reduce the charge for LCSD follows quite an opposite trend. The presence of ammonium salt in the acidic solution was found to increase the charge of LCSD. The simple mass spectrum with a narrow distribution of charge state obtained with perchloric acid at pH 1 was demonstrated to facilitate the counting of basic sites.

Effect of Terminal Phosphate Groups on Collisional Dissociation of RNA Oligonucleotide Anions.

The increasing need for mass spectrometric analysis of RNA molecules calls for a better understanding of their gas-phase fragmentation behaviors. In this study, we investigate the effect of terminal phosphate groups on the fragmentation spectra of RNA oligonucleotides (oligos) using high-resolution mass spectrometry (MS). Negative-ion mode collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) were carried out on RNA oligos containing a terminal phosphate group on either end, both ends, or neither end. We find that terminal phosphate groups affect the fragmentation behavior of RNA oligos in a way that is dependent on the precursor charge state and the oligo length. Specifically, for precursor ions of RNA oligos of the same sequence, those with 5'- or 3'-phosphate, or both, have a higher charge state distribution and lose the phosphate group(s) in the form of a neutral (H3PO4 or HPO3) or an anion ([H2PO4]- or [PO3]-) upon CID or HCD. Such a neutral or charged loss is most conspicuous for precursor ions of an intermediate charge state, e.g., 3- for 4-nt oligos or 4- and 5- for 8-nt oligos. This decreases the intensity of sequencing ions (a-, a-B, b-, c-, d-, w-, x-, y-, z-ions) and hence is unfavorable for sequencing by CID or HCD. Removal of terminal phosphate groups by calf intestinal alkaline phosphatase improved MS analysis of RNA oligos. Additionally, the intensity of a fragment ion at m/z 158.925, which we identified as a dehydrated pyrophosphate anion ([HP2O6]-), is markedly increased by the presence of a terminal phosphate group. These findings expand the knowledge base necessary for software development for MS analysis of RNA.

Charge Detection Mass Spectrometry Reveals Conformational Heterogeneity in Megadalton-Sized Monoclonal Antibody Aggregates.

Aggregation of protein-based therapeutics can occur during development, production, or storage and can lead to loss of efficacy and potential toxicity. Native mass spectrometry of a covalently linked pentameric monoclonal antibody complex with a mass of ∼800 kDa reveals several distinct conformations, smaller complexes, and abundant higher-order aggregates of the pentameric species. Charge detection mass spectrometry (CDMS) reveals individual oligomers up to the pentamer mAb trimer (15 individual mAb molecules; ∼2.4 MDa) whereas intermediate aggregates composed of 6-9 mAb molecules and aggregates larger than the pentameric dimer (1.6 MDa) were not detected/resolved by standard mass spectrometry, size exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), or by mass photometry. Conventional quadrupole time-of-flight mass spectrometry (QTOF MS), mass photometry, SEC, and CE-SDS did not resolve partially or more fully unfolded conformations of each oligomer that were readily identified using CDMS by their significantly higher extents of charging. Trends in the charge-state distributions of individual oligomers provides detailed insight into how the structures of compact and elongated mAb aggregates change as a function of aggregate size. These results demonstrate the advantages of CDMS for obtaining accurate masses and information about the conformations of large antibody aggregates despite extensive overlapping m/z values. These results open up the ability to investigate structural changes that occur in small, soluble oligomers during the earliest stages of aggregation for antibodies or other proteins.

A Gated TIMS FTICR MS Instrument to Decipher Isomeric Content of Complex Organic Mixtures.

Modern research faces increasingly complex materials with a constant need for new analytical strategies that can provide deeper levels of chemical insight. Ultrahigh resolution mass spectrometry (MS), particularly Fourier transform ion cyclotron resonance (FTICR) MS, has provided a robust analytical foundation. However, MS alone offers limited structural information. Here, we present the first implementation and results from an FTICR MS with fully integrated dual accumulation analysis with gated trapped ion mobility spectrometry (gTIMS) capability. The drastically extended charge capacity and parallel accumulation facilitate the analysis of complex mixtures. We achieved a high dynamic range of 4 orders of magnitude within a single FTICR acquisition event. Simultaneously, the valuable linear relationship between the TIMS elution voltage and reduced mobility was retained over a wide mobility range. Benchmarking the instrument performance with Suwannee River fulvic acid (SRFA) by variable ramp gTIMS analysis allowed separation and unambiguous assignment of different charge state distributions. Application to bio-oils has proven the capability to distinguish the isomeric diversity in these ultracomplex samples, while maintaining the expected FTICR MS resolving power and mass accuracy. Valuable information about the molecular distribution, isomeric diversity, and main molecular differences could directly be extracted within the analysis time of a classical "dilute and shoot" direct infusion experiment. The development of this fully integrated and flexible gTIMS with FTICR MS analysis possesses the potential to significantly change the current landscape of high-resolution mass spectrometric analysis of complex mixtures through the added insight of isomeric complexity afforded by TIMS. The exploration of the added IMS dimension promises transformative effects across diverse fields including energy transition, environmental studies, and biological research.

Investigations on MoS2 plasma by infra-red pulsed laser irradiation in high vacuum

MoS2 targets were irradiated by infra-red (IR) pulsed laser in a high vacuum to determine hot plasma parameters, atomic, molecular and ion emission, and angular and charge state distributions. In this way, pulsed laser deposition (PLD) of thin films on graphene oxide substrates was also realized. An Nd:YAG laser, operating at the 1064 nm wavelength with a 5 ns pulse duration and up to a 1 J pulse energy, in a single pulse or at a 10 Hz repetition rate, was employed. Ablation yield was measured as a function of the laser fluence. Plasma was characterized using different analysis techniques, such as time-of-flight measurements, quadrupole mass spectrometry and fast CCD visible imaging. The so-produced films were characterized by composition, thickness, roughness, wetting ability, and morphology. When compared to the MoS2 targets, they show a slight decrease of S with respect to Mo, due to higher ablation yield, low fusion temperature and high sublimation in vacuum. The pulsed IR laser deposited MoS x (with 1 < x < 2) films are uniform, with a thickness of about 130 nm, a roughness of about 50 nm and a higher wettability than the MoS2 targets. Some potential applications of the pulsed IR laser-deposited MoS x films are also presented and discussed.

Elucidating important factors and corresponding method optimization for sensitive detection of small peptide drugs in human urine by solid-phase extraction and UPLC-HRMS: The influence of MS scan modes, protein precipitants, and ammonium formate.

Small peptide hormones are widely used in sports as performance-enhancing substances, making it crucial to develop sensitive analytical methods for their detection in doping control analysis. Various factors significantly affect analytical sensitivity, such as the selection of ultra-performance liquid chromatography (UPLC) mobile phase, high-resolution mass spectrometry (HRMS) scanning modes, and extraction solvents for pretreatment. Herein, comparative study approach was utilized to investigate the sensitivity of each peptide analyte under both full scan and parallel reaction monitoring (PRM) modes of HRMS and assess the effects of some protein precipitants as a part of extraction solvents on solid-phase extraction (SPE). The results showed that full scan should be selected as the primary scan mode of HRMS, and the combination with PRM mode could effectively compensate for the limitations of full scan, and the addition of protein precipitants would adversely affect the detection of certain small peptide analytes. Meanwhile, influences of ammonium formate in reverse UPLC mobile phase on the charge state distribution of small peptides were investigated and elucidated. Based on these findings, a sensitive and reliable UPLC-HRMS analytical method combining full scan and PRM mode was validated for screening and confirmation of 63 small peptide analytes after SPE, with limits of detection (LODs) ranging between 0.010 and 0.473 ng/ml and limits of identification (LOIs) ranging between 0.015 and 1.512 ng/ml. Additionally, suggestions were provided for the detection of [Arg8]-vasopressin, dermorphin, and its analogues.

Characterization of Complex Proteoform Mixtures by Online Nanoflow Ion-Exchange Chromatography-Native Mass Spectrometry.

The characterization of proteins and complexes in biological systems is essential to establish their critical properties and to understand their unique functions in a plethora of bioprocesses. However, it is highly difficult to analyze low levels of intact proteins in their native states (especially those exceeding 30 kDa) with liquid chromatography (LC)-mass spectrometry (MS). Herein, we describe for the first time the use of nanoflow ion-exchange chromatography directly coupled with native MS to resolve mixtures of intact proteins. Reference proteins and protein complexes with molecular weights between 10 and 150 kDa and a model cell lysate were separated using a salt-mediated pH gradient method with volatile additives. The method allowed for low detection limits (0.22 pmol of monoclonal antibodies), while proteins presented nondenatured MS (low number of charges and limited charge state distributions), and the oligomeric state of the complexes analyzed was mostly kept. Excellent chromatographic separations including the resolution of different proteoforms of large proteins (>140 kDa) and a peak capacity of 82 in a 30 min gradient were obtained. The proposed setup and workflows show great potential for analyzing diverse proteoforms in native top-down proteomics, opening unprecedented opportunities for clinical studies and other sample-limited applications.

Effect of solenoidal magnetic field on ion charge state distribution in laser ion source

Applying a solenoidal magnetic field to a laser produced plasma is a method to increase the ion beam current supplied from a laser ion source to applications. The purpose of this study is to investigate the effects of the solenoid magnetic field on the charge state distribution of the ion beam extracted from the laser ion source. A plasma generated by a Nd: YAG laser was injected into the solenoid lens with a length of 25 mm. The current waveforms of ion beams extracted from the laser produced plasmas were measured as a function of solenoidal field strength. In addition, the variation of beam current waveforms for each charge state ion was investigated. The results indicated that the charge state distribution in the plasma was kept after focusing by the solenoid magnetic field.

Radioactive ion-beam development at SPIRAL1

SPIRAL1 at GANIL is a facility dedicated to the production of radioactive ion beams (RIBs) and their post-acceleration. A major upgrade allowed this facility to produce RIBs of condensable elements. It does so by using both a hot 1+ ion source and an ECR Charge Breeder where the latter is used to increase the ion charge state for post-acceleration. The SPIRAL1 team is pursuing R&D on both fronts in order to expand the offer of RIBs. Several 1+ sources have been or will be tested in 2023: (1) MonoNaKe, a surface ion source, is being modified to ionize short-lived lithium isotopes (2) TULIP, dedicated to ionize short lived fusion-evaporation residues has been tested online for the second time (3) the FEBIAD source has produced radioactive Cr ions for the first time. The SPIRAL1 Charge Breeder (SP1CB) is now fitted with a fixed frequency amplifier (Klystron) and a variable frequency amplifier (travelling wave tube). Using one amplifier, the other, or both, enables several heating modes, giving better control on the charge state distribution at the output of SP1CB.

Upgrade and Improvement of the TRIUMF ECRIS Charge State Booster

Recently, the RF system of the TRIUMF electron cyclotron resonance ion source charge state booster (ECRIS CSB) underwent an upgrade to implement two-frequency heating using a single waveguide. The injection and extraction optics, as well as the injection and extraction systems, were carefully modelled and systematically optimized to improve the efficiency and beam quality of the charge state booster. With optimized plasma and beam optics under the single-frequency heating regime, the maximum charge state of the 133Cs isotope produced was 27+, with the peak of the charge state distribution at Cs23+ with an efficiency of 8.5 %. However, with the implementation of two-frequency heating, the maximum charge state of Cs that can be produced increased to 32+, and the charge state distribution’s peak shifted to Cs26+ with an efficiency of 9.1 %. Additionally, the two-frequency heating resulted in a total beam RMS emittance that was approximately half of the one that was measured under the single-frequency heating due to the more pronounced negative potential dip created at the plasma center.

Application of Optical Emission Spectroscopy to Electron Cyclotron Resonance Ion Sources

Electron Cyclotron Resonance Ion Sources (ECRIS) are widely used for production of highly charged high intensity ion beams for research, medical and industrial applications.ECRIS performances, especially the charge state distribution and beam intensity, depend significantly on the electron energy distribution function that ranges from a few eV to hundreds of keV. Further improvements of ECRIS performances require a deeper and deeper understanding of the plasma heating mechanisms and ion generation by means of opportune plasma diagnostics. Amongst others, optical emission spectroscopy (OES) is the most remarkable for application in ECRIS: it is a non-invasive diagnostics able to operate also in high-voltage conditions and it requires small room for operation. OES has been already tested for plasma diagnostics in proton sources.This work presents the experimental set-up developed for the plasma diagnostics of the Advanced Ion Source for Hadrontherapy (AISHa), an ECRIS for medical applications, together with the strategy applied to relate plasma emission lines in the visible and near-infrared domain to plasma parameters for some ions of interest. Preliminary results and perspectives will be also discussed.

Dual Frequency Enhancement of the Supernanogan Multi-Charged Ion Source at TRIUMF ISAC Facility

In 2008, a Supernanogan ECR ion source from PANTECHNIK was introduced in addition to an in-house developed microwave ion source and a surface ion source to complement the TRIUMF Offline Ion Source (OLIS) facility to provide highly charged ions to ISAC experiments. Originally, it employed a 400 W Travelling Wave Tube Amplifier (TWTA) for RF heating, but less than 50 W was enough to produce all the multi-charged beams required by the experiments. A 50 W solid-state amplifier was added for redundancy purposes, but we found a significant improvement when both were switched on at the same time. When properly optimized for the dual frequency, less than ten times the total power is needed to produce a similar charge state distribution with more current than the single frequency. The beam stability and the ability to extract higher charged ions also improved with the dual frequency enhancement. The simulation studies, operational experience, and results are discussed in this paper.

Enhanced Production of Multicharged Ions by Mixing Low Z Gas and Emittance Measurement on Electron Cyclotron Resonance Ion Source

We have studied to enhance efficiency of producing multicharged ions in an electron cyclotron resonance ion source (ECRIS). The gas mixing method is known as an empirical method for highly efficient production of multicharged ions. This method is to mix low Z gas into the multicharged ions plasma. There are various explanations for the gas mixing effect. One of them is the ion cooling effect. It is necessary to obtain parameters related to the ion temperature in order to experimentally confirm the effect. We measured emittances for ion beams by using a wire probe and a multi-slit. He was mixed as a low Z gas into the operating Ar gas. Charged state distributions of ion beams was measured to confirm the gas mixing effect. We have also measured emittances for the multicharged Ar ion beams and compared root mean square emittances (ε rms) of those for pure Ar and Ar mixed by He. We confirmed that ε rms for Ar mixed by He was lower than that for pure Ar.