Tandem Mass Spectrometry of Peptide Ions by Microwave Excited

Subscriber access provided by Kaohsiung Medical University

Article

Tandem Mass Spectrometry of Peptide Ions by Microwave Excited Hydrogen and Water Plasmas Yuji Shimabukuro, Hidenori Takahashi, Shinichi Iwamoto, Koichi Tanaka, and Motoi Wada Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00344 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Tandem Mass Spectrometry of Peptide Ions by Microwave Excited Hydrogen and Water Plasmas Yuji Shimabukuro*,†, Hidenori Takahashi‡, Shinichi Iwamoto‡, Koichi Tanaka‡, and Motoi Wada† †Graduate School of Science and Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan ‡Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu Corporation, Nakagyo-ku, Kyoto 604-8511, Japan KEYWORDS: Mass Spectrometry, Inductively Coupled Plasma, Capacitively Coupled Plasma, Microwave Plasma, Radical Induced Dissociation ABSTRACT: A thermal cracking cell that served as the atomic hydrogen source for hydrogen attachment/abstraction dissociation (HAD) analysis has an intrinsic problem to produce a beam of atoms reactive against heated tungsten capillary. A plasma excited by 2.45 GHz microwave discharge can deliver reactive species to a quadrupole ion trap confining analyte ions without excessive heating of the radical source components. The radical (H•) production performance of the developed source was evaluated by optical emission spectroscopy and H• attachment reaction to fullerene ions. The source exhibited the H• attachment rate as high as a thermal cracking source forming H• in the high temperature tungsten capillary to induce fragmentation processes preserving posttranslational modifications. Water vapor was introduced to the source to confirm the stability to generate oxygen containing radicals, which were found present in the water vapor plasma together with atomic hydrogen. Injection of radicals from a water vapor plasma successfully dissociated peptide ions to c-/z- and a-/x- type ions as the case of HAD induced by a thermal cracking cell.

INTRODUCTION Proteome analyses using radical-induced dissociation (RID)a is advantageous over the conventional low-energy collisioninduced dissociation (LE-CID)1,b in tandem mass spectrometry (MS/MS) c , especially for post-translational modifications (PTMs)d and top-down proteome analyses. The commercially available RID-MS/MS of electron capture dissociation (ECD)2,3,e and electron transfer dissociation (ETD)4,f have been widely applied for the practical analysis in complex biological mixtures. However, ECD/ETD are only applicable to the dissociation of multiply charged ions. Our research group has developed RID of hydrogen attachment/abstraction dissociation (HAD)5,g which can be applied to any charge state of ions. Since HAD employs neutral hydrogen radicals for the dissociation, the neutralization loss of the precursor ions can be avoided during the dissociation process. To extend the original concept of HAD, gas-phase radicals produced from water and oxygen molecules were employed for the dissociation of peptide ions in this study. In our previous HAD study, hydrogen radicals were formed in a high temperature tungsten capillary tube heated to 2,300 K by a tung-

sten filament. The high temperature components of this type of thermal cracking source do not only cause an outgassing from heated components but also has a short operational lifetime as tungsten becomes reactive against oxygen and halogens at an elevated temperature. A radio frequency (RF) h plasma source can dissociate many kinds of reactive gases such as oxygen, water vapor, hydrochloric acid, etc. and is suitable as a radical source for operation with chemically reactive species. The RF plasma generator neither has high temperature components nor requirements to arrange complicated electrode structures to maintain high electric current required to run a heater in vacuum. The electrode structure of the source determines the discharge modes; the RF plasma are distinguished by plasma density into capacitively coupled plasma (CCP)i and inductively coupled plasma (ICP)j.6 In general, the radio frequency such as 13.56, 27.12, or 40.68 MHz are utilized for RF plasma. However, MHz frequency requires higher operating pressure (~101 Pa) and higher input level (~102 W)7,8 to ignite and sustain a plasma. Our previous work indicated that the electrode geometry to enhance the local electric field was an important factor to reduce the operation gas pressure and increase the degree of dissociation with 2.45 GHz microwave driven CCP atomic sources.9 Further study on atomic source using 2.45 GHz microwave plasma excitation is being made, and we report the

a

RID: Radical Induced Dissociation LE-CID: Low-Energy Collision-Induced Dissociation c MS/MS: tandem mass spectrometry d PTM: Post-Translational Modification e ECD: Electron Capture Dissociation f ETD: Electron Transfer Dissociation g HAD: Hydrogen Attachment/Abstraction Dissociation b

h

RF: Radio Frequency CCP: Capacitively Coupled Plasma j ICP: Inductively Coupled Plasma i

1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

HAD performance of an electrodeless discharge system, which we call ECR k -ICP (electron cyclotron resonance-inductively coupled plasma) source. An optical emission spectroscopy confirmed the presence of radical species in the discharge plasma for both CCP and ECR-ICP configuration. The transport of radicals reaching from the discharge region to the reaction chamber is confirmed by the atom attachment reaction to fullerenes (C60)10.

Page 2 of 21

were mixed on a MALDI sample target well. Powdery C60 was dissolved in 100% toluene, and the solution (1 µL) was spotted on the MALDI sample target well. ECR-ICP Radical Source Design Figure 1 schematically shows the structure of a spiral antenna type microwave driven ECR radical source. This ECR-ICP radical source can be mounted to a ConFlatⓇ 70 flange. A water-cooled toroidal Sm-Co magnet (φ22×φ18×32) mounted in the source tip forms a magnetic field structure with the region of the intensity greater than 875 G corresponding to the ECR at 2.45 GHz along a quartz glass capillary to achieve a higher plasma density. A plasma is ignited in the 119 mm long 6.5 mm inner diameter quartz glass capillary through coupling the microwave power by a 15-turns 0.3 mm thick copper ribbon spiral antenna coiled around the capillary. The gas of the target element is directly injected into the capillary. The radicals produced in the plasma travel toward the quadrupole ion trap passing through the source orifice shown in Figure 1 located at 131.5 mm from the 1.5 mm-diameter aperture in the ring electrode of the ion trap. A 3 mm diameter aperture opened on a stainless-steel source orifice reduces the radius of the radical beam from the plasma. Microwave power is applied to the copper spiral antenna through a coaxial transmission line via N-type RF feedthrough (Cosmotec, C34NR1). A 3.75 mm diameter center copper rod, an alumina insulator, and a 6 mm inner diameter 8 mm outer diameter copper shield tube form a coaxial transmission line from the RF feed through to the spiral antenna. Ceramic coatings are applied to the both ends of the coaxial transmission line to enhance the voltage holding between the center and outer copper electrodes. The coaxial line has 9.7 Ω characteristic impedance while the transmission impedance of the coaxial cable from the microwave power supply is 50 Ω. Thus, the microwave reflection back to the power supply is reduced by inserting a triple stub-tuner between the cable and the N-type feedthrough.

EXPERIMENTAL SECTION To realize a RID/MS system a microwave driven radical source was attached to a quadrupole ion trap (QIT)11,l coupled to a time of flight (TOF)m mass analysis system. In this section, the tandem mass spectrometer for HAD study and the detail of the microwave driven ECR-ICP radical source are described. RID Experimental Setup All mass analyses were performed on the prototype MALDInQIT-TOF tandem mass spectrometer based upon the design of the AXIMA Resonance (Shimadzu/ Kratos). The developed ICP radical source is directly mounted outside of the ring electrode of the QIT chamber. The ring electrodes of the QIT chamber has two 1.5 mm diameter holes for injecting radicals from the source. A nitrogen laser (wavelength: 337 nm) ionizes a target material by a MALDI12-14 system. The ionized target passes through the end cap electrode and is trapped in the QIT chamber. Helium gas cools the trapped ions, while argon gas induces conventional collision induced dissociation (CID)o. The reaction time between injected radical and target ions is set from 0.5 to 30 s. The pressure inside the QIT chamber is maintained below 5×10-4 Pa before the radical injection. Digital Asymmetric Wave Isolation (DAWI)15, p confines the precursor ions in the QIT, and a time of flight (TOF) mass spectrum of the detected fragment ions is recorded with 10 kV accelerating voltage in the reflectron mode. Mass spectra were acquired by averaging 50-100 single shots. Oxygen gas was introduced by pressure regulator (GL Sciences, CR-10-2, Tokyo, Japan). A needle valve (2400T, Kofloc, Kyoto, Japan) controlled the hydrogen, oxygen and water vapor flow rates to the ECR-ICP source. Material Preparation Sample solution was dissolved in 30% acetonitrile (ACN) /0.1% aqueous trifluoroacetic acid (TFA) (v/v). α-cyano-4hydroxycinnamic acid (CHCA) matrix was dissolved at a concentration of 10 mg/mL in 50% ACN/0.1% aqueous TFA (v/v). Peptide standards, fullerene C60 powder and MALDI matrix were purchased from Sigma-Aldrich (St. Louis, MO), Ana Spec Inc. (San Jose, CA, USA) and Peptide Institute (Osaka, Japan). Sample solution (0.5 µL) and matrix solution (0.5 µL)

Figure 1. A schematic diagram of the RID experimental setup and sectional view of the ECR-ICP source. The MALDI-QIT-TOF mass spectrometer equips the ECR-ICP radical source. A 2.45 GHz microwave power is matched by the triple stub tuner.

k

ECR: Electron Cyclotron Resonance QIT: Quadrupole Ion Trap m TOF: Time-Of-Flight n MALDI: Matrix Assisted Laser Desorption/Ionization o CID: Collision-Induced Dissociation p DAWI: Digital Asymmetric Wave Isolation

RESULTS and DISCUSSION Radical Source Performance A microwave ECR-ICP was successfully formed in the quartz capillary along the inner wall. An inductive coupling antenna for a plasma source generally excites two types of discharge modes: E (Electrostatic) mode and H (electromagnetic) mode.

l


Page 3 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The ICP source started an ignition with E mode discharge and showed the mode transition to H mode increasing the electron density with a brighter plasma glow.6 Both large input power and proper matching by the tuning stubs were indispensable for the mode jump. After the mode jump was established the source operated with H mode with a reduced microwave input power. All experiments reported here were carried out with the H mode discharge, or the condition realized after the mode jump. All gas species show the ignition characteristics similar to the lower pressure side of the Paschen valley. The ECR-ICP source can sustain a stable discharge under the 10-1 Pa pressure and 20 W input power conditions. Lower input power under 20 W provided a faint emission of the E mode discharge condition. The E mode maintained the discharge as low as 5 W microwave power input. A power input higher than 20 W at an elevated pressure recovers an H mode plasma from E mode, which can be easily confirmed with substantial increase in brightness. Optical Emission Study of Hydrogen Plasmas The microwave radical source was attached to a separate vacuum chamber having a cross port geometry connecting the radical source, two glass view ports, a Pirani gauge (ULVAC, GP-2A) and a vacuum ionization gauge (ULVAC, GI-TL3) to a turbomolecular pump (Pfeiffer, TMH262). The chamber is made of stainless steel with the size of 70 mm on each side. The glass view port attached in front of the radical source and that attached sideway to the source axis enable observations of the plasma glow from the corresponding directions. Optical emission spectrum (OES) q gives a rough estimate on what kind of excited species are present in the plasma. Optical fibers fixed at the center of the view port facing the ECR-ICP source orifice deliver optical signals to the two optical multichannel analyzers to investigate the OES in both visible light region (Ocean optics USB 2000+), and ultraviolet region (Ocean optics Flame-S), respectively. Figure 2 shows the typical OES from the ECR-ICP and CCP9 for hydrogen. Since the signal depends on the size of the aperture, gas pressure and matching condition, the ordinates do not coincide among different spectra. Each gas pressure and input power are adjusted following the experimental condition for HAD. Hydrogen gas ECR-ICP and CCP spectra in the visible range indicate the presence of excited hydrogen atoms and molecules. The brightest two peaks correspond to the atomic hydrogen spectra of Balmer alpha (656 nm) and beta (486 nm) lines. A decrease in hydrogen gas pressure and an increase in the microwave power reduced the molecular emission spectra in the wavelength range from 500 nm to 800 nm. These spectra called Fulcher band are typical optical emission from hydrogen molecules due to their rotational and vibrational excitations.16 We do not put hydrogen spectra of ultraviolet region, because, in the ultraviolet region, the hydrogen spectra do not show sufficient intensity of atomic emission compared to background molecular spectra.

q

Figure 2. Optical emission spectra of two types of microwave hydrogen plasmas measured by USB 2000+. (A) H mode hydrogen ECR-ICP, 1.4×10-1 Pa, 56.7 W. (B) hydrogen CCP, 1.3 Pa, 100W.

Hydrogen Attachment to Fullerenes Following Demirev’s experiment of H radical (H•) production in FT-ICR10, r, relative H radical density inside the ion trap was estimated by observing H radical attachment to fullerene ions. Figure 3 show the isotopic distribution of C60+• observed at 1.0 s of reaction time using (A) ICP discharge, (B) CCP discharge9, and (C) thermal cracking used in our previous work of HAD5 operated with pure H2 gas. The flow rates of input H2 gas are set to 4 sccm in thermal cracking source and 100 sccm in microwave plasma source. The H• attachments roughly from 10 to 20 were observed for all H radical sources, indicating that the H• were efficiently transported into the ion trap. The degree of dissociation of thermal cracking source can be estimated below 50 %5,17. Meanwhile, the cracking efficiency of the ECR-ICP and CCP sources appear lower than the thermal cracking source as they require higher H2 flow rates. However, the ECR-ICP source achieves the highest number of H• attachment to fullerenes among the three sources in the same reaction time. Although the attachment efficiency cannot be directly compared at different source operation pressure, the efficiency for H• radical transport from the ECR-ICP and CCP sources to fullerene ions in QIT can be estimated comparable to the thermal cracker source that demonstrated the HAD reaction.

r

OES: Optical Emission Spectroscopy

FT-ICR: Fourier Transform Ion Cyclotron Resonance



Page 4 of 21

3 -1 s for hot H radicals with their energy exceeding 1 eV, and the rate constant is decreased by 6 orders of magnitude at room temperature.18 Density functional theory (DFT)s calculation done by Asakawa et al. shows that cleavage of the N-Cα bond by H radical attachment requires the radical to possess enough kinetic energy; there is a threshold in reaction. 19 As we will show in the next section from Hα/Hβ ratio, effective temperature of CCP is expected higher than that of ECR-ICP. It should be this difference in effective temperature that makes the reaction for ECR-ICP less efficient.

Figure 3. Product ion spectra of H• attachments to fullerene ion C60+• for 1.0 s reaction time using (A) ECR-ICP with H2 gas (B) CCP with H2 gas, and (C) thermal cracking source.

Dissociation of Peptide Ions by Hydrogen Plasma To demonstrate the dissociation of peptide ions using microwave driven radical sources, 1+ substance P (RPKPQQFFGLM-NH2, 1347 Da) in QIT was exposed to H radical irradiation generated by ECR-ICP and CCP of H2 gas (Figure 4). The fragmentation due to H attachment, H abstraction, or any other process by ECR-ICP was unobserved even if the reaction time was increased up to 30 s (data not shown), while the 2+ precursor ions corresponding to 674 of m/z were observed. On the other hand, in the hydrogen CCP, the abundant c-/z- and a-/x- type ions attributable to radical induced dissociation of the peptide backbone were clearly observed. The peak of 2+ ions were also observed with 20 % of relative intensity. These results can suggest that the kinetic energy, or “the effective temperature” of H radicals generated by ECRICP is not high enough to react with peptide ions. Tureček and co-workers reported that the rate constant of direct H attachment to amide carbonyl groups is on the order of 10-12 cm-

Figure 4. Fragmentation spectra of singly protonated substance P obtained by (A) ECR-ICP of H2 gas: 5 s, (B) CCP of H2 gas: 1s, and (C) thermal cracking of H2 gas: 0.5s.

In the pure hydrogen CCP, a lot of a- type product ions are generated in addition to one of the original HAD by the thermal cracking source. According to the OES result shown in Figure 2 (A) and (B), hydrogen CCP contains vibrationally excited hydrogen molecules, hydrogen ions, and probably electrons other than H radicals, while the thermal cracking source mainly produces the hydrogen atoms due to its catalytic effect on the heated tungsten capillary. Since the present microwave driven radical source is not equipped with electron deflection magnets at the source due to space requirements,

s

DFT: Density Functional Theory


Page 5 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry Figure 5. Optical emission spectra of microwave water ECR-ICP measured by USB 2000+ and Flame-S at 9.5×10-2 Pa, 65.0 W. (A) Visible light region. (B) Ultraviolet region.

substantial number of electrons can penetrate the ion trap compare to the thermal cracking source that has electron deflection magnets.

OES of Water Plasmas Figures 5 and 6 show the optical emission spectra of a water plasma produced by ECR-ICP and that by CCP, respectively. Water plasmas contain H•, O, and hydroxyl radicals (OH•). The emission signal from OH• radical is observed in ultraviolet region at 309 nm (A2Σ+-X2Πi).20 In addition, the water plasma shows atomic hydrogen spectra up to Balmer gamma and delta lines (434 and 410 nm) which are the higher excited levels of hydrogen atoms. The Balmer alpha to Balmer beta intensity ratio (Hα/Hβ) can give rough estimate on the electron temperature since the higher excited species are preferentially excited in a high temperature plasma. The value was 3.6 for a pure hydrogen ECRICP. Frantz and Wünderlich gave the relationship between line ratio (Hα/Hβ) and electron temperature in the case of a pure hydrogen discharge.21 According to their data, the line ratio from 2.2 to 3.6 corresponds to about 2 to 4 eV of electron temperature with the larger intensity ratio corresponding to lower electron temperature. Meanwhile the measured ratio was 2.9 for water ECR-ICP with the values being 3.1 and 2.2 for hydrogen CCP and water CCP, respectively. These values indicate that the water plasma contains more highly excited species compared to a pure hydrogen plasma. The active species formed in a water vapor plasma may also have higher electron temperature compared to a pure hydrogen plasma. This postulation also agrees with the general understanding that CCP plasma has a higher effective temperature than ICP plasma.22

Figure 6. Optical emission spectra of microwave water CCP measured by USB 2000+ and Flame-S at 4.2×10-1 Pa, 26.6 W. (A) Visible light region. (B) Ultraviolet region.

Attachment of Water Vapor Plasma Radicals to Fullerenes To confirm the production and transport of reactive species formed in the water plasma produced by ECR-ICP and CCP sources, radicals were directed to the QIT confining fullerene ions. The attachment spectra obtained by the water vapor discharge of microwave plasma sources are shown in Figure 7. Radical attachments at higher masses are observed compared with pure hydrogen discharge shown in Figure 3. A bold line drawn in the 1 s spectrum is the smoothed spectrum obtained by a binomial filter. Unlike the case of pure hydrogen discharge, the time evolution spectra show two groups. It was also confirmed that the pure oxygen discharge by ECR-ICP source realized attachment reaction to C60+• (data not shown). Since the mass difference between the first and the second peak groups to compare in Figure 7 show separation of 16 Da, O or OH• in the water vapor plasma seems to cause these characteristic mass spectra with two peak-groups. However, which reactive species is/are responsible for these unique spectra cannot be specified at this moment except that a water plasma can produce at least two kinds of reactive species. Dissociation of Peptide Ions by Water Plasma Water plasmas containing H•, OH• and/or O were produced by ECR-ICP and CCP to see the effect of the produced particle



Page 6 of 21

ECR-ICP of H2O vapor and (B) CCP of H2O vapor.

flux onto dissociation of 1+ substance P in the QIT. Figure 8 (A) and (B) show the product ion spectra of 1+ substance P using ECR-ICP and CCP of H2O, respectively. The reaction times were set from 0.5 s to 1.0 s. These spectra also showed abundant c-/z- and a-/x- type ions, as well as attachments of O (+16) and water molecules (+36). As a comparison, Figure 4 (C) shows the product ion spectrum of substance P using thermal cracking source. While the relative intensity of product ions by the thermal cracking hydrogen source is higher than water plasmas by an order of magnitude, the types of product ions are similar to those of water vapor plasmas. This result indicates that H•, OH•, O, and/or excited molecules mixture generated by water vapor plasmas causes the radical induced dissociation similar to HAD by a thermal cracker source.

Figure 8. Fragmentation spectra of singly protonated substance P obtained by (A) ECR-ICP of H2O vapor and (B) CCP of H2O vapor.

The charge enhanced 2+ precursor ions of m/z 674 are observed in all spectra obtained by microwave plasma sources. This can be possibly due to the electron ionization of the 1+ precursor ions as discussed in the section of hydrogen operation. To identify the cause of these 2+ precursor ions, electron beam injection without any discharge was carried out. Figure 9 shows the spectrum obtained by injecting an electron beam generated by thermal electron emission from a hot tungsten filament installed at 50 mm away from the inlet hole opened on the ring electrode of the QIT. The 1+/2+ product ions and a lot of charge enhanced 2+ precursor ions are observed like the case of electron ionization dissociation.23 From this observation, the main factor of 2+ precursor ion production mechanism is speculated as electron injection to the ion trap from the microwave source. There still remains, however, the possibility for other charged and/or excited species entering the system to produce 2+ precursor ions.

Figure 9. Fragmentation spectra of singly protonated substance P obtained by electron beam injection.

Source Operation with Pure O2 The product ion spectra by reactive species injection from microwave plasma sources are observed different from those obtained with thermal cracking source. The kinds of product ions generated by water plasma injection are almost identical with the original HAD spectrum shown in Figure 4 (C). Thus, these product ion spectra are probably caused by H attachment and/or abstraction to/from precursor ions like the case of HAD. Since a water plasma mainly contains H•, OH•, and O according to the OES results shown in Figure 5 and 6. A pure oxygen discharge was run by attaching the microwave radical source to the HAD setup for confirming the effect of oxygen atoms. Oxygen plasma was produced with ECR-ICP as CCP plasma has a tungsten electrode in contact with plasma, which should contaminate the mass data by tungsten atoms. An OES obtained for oxygen discharge is shown in Figure 10 with three prominent atomic oxygen line spectra at 777.4, 844.6, 926.6 nm and the first negative system of O2+ ions (b4 Σ -a4 Π u).24 Figure 11 shows the product ion spectrum obtained by pure oxygen ECR-ICP for singly protonated substance P. While oxidized precursor ions were clearly observed in the spectrum, the product ions were predominantly oxidized

Figure 7. Product ion spectra of some reactive species attachments to fullerene ion C60+• for 0.25 and 1.0 s reaction times using (A)


Page 7 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a- type ions (a•+16Da) which were not observed for a water vapor plasma. This result, as compared with Figure 8, suggests that the atomic oxygen does not play an important role in dissociation by water plasma based radical injection. Thus, the fragmentation caused by OH• may exist, while it can be affected due to the presence of H•.

like fragmentation of substance P with both hydrogen and water plasmas. The effective temperature of radicals may affect the dissociation process of peptide ions. The 2+ precursor ions in the HAD spectrum obtained by water CCP was found lower as compared to other a-type product ions. From the view point of hardware operation, the CCP source does not need complicated matching procedure like the ECR-ICP source. The microwave radical source can be further optimized for RID analyses by resolving problems like the electron drain into QIT causing part of 2+ precursor ions.

Figure 10. Optical emission spectrum of microwave oxygen ECRICP measured by USB 2000+ at 1.4×10-1 Pa, 85.0 W.

Figure 12. M+• ratio with respect to original precursor [M+H]+ intensity when changing the H2O partial pressure with H2 pressure.

CONCLUSION The realization of HAD fragmentation utilizing a microwave plasma source with MALDI-produced protonated peptide ions was reported. To achieve an extension of the RID applications, radical sources based on the microwave excited ECR-ICP and CCP plasma source have been developed. The production and transport efficiencies of the reactive agents generated by the microwave sources were confirmed comparable to those by a thermal cracking source. A series of experiments has shown that CCP source produces a plasma of a higher effective temperature appropriate for peptide analysis that requires higher reaction energy. Meanwhile the ECR-ICP source with the lower effective temperature plasma will be useful for other reaction analyses that requires lower effective temperature. The HAD system equipped with water CCP source has a potential of replacing the original HAD which employs a hydrogen gas supply system. Electrodeless discharges maintained in ECR-ICP will allow one to introduce highly reactive chemical gas for analysis. The RID based upon the injection of wide variety of radicals from various kinds of chemicals will enable the expansion of the types of reaction and kind of targets.

Figure 11. Fragmentation spectra of singly protonated substance P obtained by pure oxygen ECR-ICP.

Comparison between H2O Plasma and H2 Plasma Water vapor can replace gaseous hydrogen injection into the system to realize HAD. Figure 8 show the H abducted precursor ions M+• in the fragmentation spectra. To evaluate the effectiveness of water injection against hydrogen, the relative intensity of abducted precursor ions (M+•) against original precursor ions ([M+H]+) was measured as the water gas pressure was changed with respect to hydrogen gas pressure. Note that the observation of H attached precursor ions ([M+H+H]+) was difficult due to the isotopic distributions. Figure 12 shows the relative intensity increases with decreasing water gas pressure; the efficiency for H abstraction from precursor ions by H• is more efficient for a hydrogen plasma operation than a water plasma operation. The water plasma seems to have 50% efficiency against hydrogen plasma, according to the figure. Although the observed intensity of product ions obtained by water CCP are lower than those of thermal cracking source, the fragmentation pattern by water CCP shown in Figure 8 (B) is a straight forward to interpret compared with thermal cracking source for c8+, c9+, and c10+ ions. There were observed a small difference in their fragmentation patterns in two kinds of microwave plasma excitation. The CCP source realized HAD

AUTHOR INFORMATION Corresponding Author * Yuji Shimabukuro. E-mail: [email protected].

Phone: +81 774 65 6349.



Page 8 of 21

(15) Brancia, F. L.; McCullough, B.; Entwistle, A.; Grossmann, J. G.; Ding. L. J. Am. Soc. Mass Spectrom. 2010, 21, 1530-1533. (16) Crosswhite, H. M. The hydrogen molecule wavelength tables of Gerhard Heinrich Dieke 1972, New York: Wiley-Interscience. (17) Schwarz-Selinger, T.; von Keudell, A.; Jacob, W. J. Vac. Sci. Tecnol., A 2000, 18, 995-1001. (18) Turecek, F.; Syrstad, E. A. J. Am. Chem. Soc. 2003, 125, 33533369. (19) Asakawa, D.; Takahashi, S.; Iwamoto, Shinichi.; Tanaka, K. Phys. Chem. Chem. Phys. 2018, DOI: 10.1039/C8CP00733K. (20) Dieke, G. H.; Crosswhite, H. M. J. Quant. Spectrosc. Radiat. Transfer. 1961, 2, 97-199.

ORCID

Yuji Shimabukuro: 0000-0003-4090-3995 Hidenori Takahashi: 0000-0001-6887-1724

Note The authors declare no competing financial interest.

(21) Frantz, U.; Wünderlich, D. New J. Phys. 2006, 8, 301/1-22. (22) Sakamoto, Y.; Maeno, S.; Tsubouchi, N.; Kasuya, T.; Wada, M. J. Plasma Fusion Res. 2009, 8, 587-590. (23) Fung, Y. M. E.; Adams, C. M.; Zubarev, R. A. J. Am. Chem. Soc. 2009, 131, 9977-9985. (24) Lock, E. H.; Fernsler, R. F.; Slinker, S.; Walton, S. G. Experimental and Theoretical Estimation of Excited Species Generation in Pulsed Electron Beam-Generated Plasmas Produced in Pure Argon, Nitrogen, Oxygen, and Their Mixtures 2011, Washington, DC: Naval Research Laboratory.

REFERENCES (1) McLuckey, S. A. J. Am. Soc. Mass. Spectrom. 1992, 3, 599-614. (2) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-3266. (3) Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N. A.; Lewis, M. A.; McLafferty, F. W. Anal. Chem. 2000, 72, 563-573. (4) Syka, J. E.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 9528-9533. (5) Takahashi, H.; Sekiya, S.; Nishikaze, T.; Kodera, K.; Iwamoto, S.; Wada, M.; Tanaka, K. Anal. Chem. 2016, 88, 3810-3816. (6) Turner, M. M.; Lieberman, M. A. Plasma Sources Sci. Technol. 1999, 8, 313-324. (7) Hopwood, J. Plasma Sources Sci. Technol. 1992, 1, 109-116. (8) Ohachi, T.; Yamabe, N.; Shimomura, H.; Shimamura, T.; Ariyada, O.; Wada, M. J. Cryst. Growth 2009, 311 2987-2991. (9) Shimabukuro, Y.; Takahashi, H.; Wada, M. Jpn. J. Appl. Phys. 2018, 57, 01AA02-1-4. (10) Demirev, P. A. Rapid Commun. Mass Spectrom. 2000, 14, 777781. (11) Paul, V. W.; Steinwedel, H. Z. Naturforschg. 1953, 8a, 448-450. (12) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T.; Matsuo, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-153. (13) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (14) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A-1203A.

for TOC only


Page 9 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. A schematic diagram of the RID experimental setup and sectional view of the ECR-ICP source. The MALDI-QIT-TOF mass spectrometer equips the ECR-ICP radical source. A 2.45 GHz microwave power is matched by the triple stub tuner. 549x197mm (72 x 72 DPI)

ACS Paragon Plus Environment


Figure 2. Optical emission spectra of two types of microwave hydrogen plasmas measured by USB 2000+. (A) H mode hydrogen ECR-ICP, 1.4×10-1 Pa, 56.7 W. (B) hydrogen CCP, 1.3 Pa, 100W. 776x956mm (72 x 72 DPI)


Page 10 of 21

Page 11 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Optical emission spectra of microwave water ECR-ICP measured by USB 2000+ and Flame-S at 9.5×10-2 Pa, 65.0 W. (A) Visible light region. (B) Ultraviolet region. 705x866mm (72 x 72 DPI)



Figure 3. Product ion spectra of H• attachments to fullerene ion C60+• for 1.0 s reaction time using (A) ECR-ICP with H2 gas and (B) CCP with H2 gas, and (C) thermal cracking source. 430x752mm (96 x 96 DPI)


Page 12 of 21

Page 13 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Fragmentation spectra of singly protonated substance P obtained by (A) ECR-ICP of H2 gas: 5 s, (B) CCP of H2 gas: 1s, and (C) thermal cracking of H2 gas: 0.5s. 705x1018mm (72 x 72 DPI)



Figure 6. Optical emission spectra of microwave water CCP measured by USB 2000+ and Flame-S at 4.2×10-1 Pa, 26.6 W. (A) Visible light region. (B) Ultraviolet region. 705x874mm (72 x 72 DPI)


Page 14 of 21

Page 15 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Product ion spectra of some reactive species attachments to fullerene ion C60+• for 0.25 and 1.0 s reaction times using (A) ECR-ICP of H2O vapor and (B) CCP of H2O vapor. 430x501mm (96 x 96 DPI)



Figure 8. Fragmentation spectra of singly protonated substance P obtained by (A) ECR-ICP of H2O vapor and (B) CCP of H2O vapor. 529x510mm (72 x 72 DPI)


Page 16 of 21

Page 17 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9. Fragmentation spectra of singly protonated substance P obtained by electron beam injection. 529x254mm (72 x 72 DPI)



Figure 10. Optical emission spectrum of microwave oxygen ECR-ICP measured by USB 2000+ at 1.4×10-1 Pa, 85.0 W. 529x360mm (96 x 96 DPI)


Page 18 of 21

Page 19 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 11. Fragmentation spectra of singly protonated substance P obtained by pure oxygen ECR-ICP. 529x254mm (72 x 72 DPI)



Figure 12. [M+H-H]+ ratio with respect to original precursor [M+H]+ intensity when changing the H2O partial pressure with H2 pressure. 340x209mm (285 x 285 DPI)


Page 20 of 21

Page 21 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC only 891x492mm (72 x 72 DPI)


Tandem Mass Spectrometry of Peptide Ions by Microwave Excited

Recommend Documents