Structural Analysis of Phospholipid using Hydrogen Abstraction

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Structural Analysis of Phospholipid using Hydrogen Abstraction Dissociation and Oxygen Attachment Dissociation in Tandem Mass Spectrometry Hidenori Takahashi, Yuji Shimabukuro, Daiki Asakawa, Shosei Yamauchi, Sadanori Sekiya, Shinichi Iwamoto, Motoi Wada, and Koichi Tanaka Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00322 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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Analytical Chemistry

Structural Analysis of Phospholipid using Hydrogen Abstraction Dissociation and Oxygen Attachment Dissociation in Tandem Mass Spectrometry

Hidenori Takahashi,1* Yuji Shimabukuro,2 Daiki Asakawa,3 Shosei Yamauchi,1 Sadanori Sekiya,1 Shinichi Iwamoto,1 Motoi Wada,2 and Koichi Tanaka1

1. Koichi

Tanaka

Mass

Spectrometry

Research

Laboratory,

Shimadzu

Corporation,

1

Nishinokyo-Kuwabaracho Nakagyo-ku, Kyoto 604-8511, Japan; 2. Graduate School of Science and Engineering, Doshisha University, 1-3 Kyotanabe, Kyoto 610-0321, Japan; 3. National Institute of Advanced Industrial Science and Technology (AIST), National Metrology Institute of Japan (NMIJ), Tsukuba Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki, 305-8568, Japan

Correspondence to: Hidenori Takahashi Koichi

Tanaka

Mass

Spectrometry

Research

Laboratory,

Shimadzu

Corporation,

1

Nishinokyo-Kuwabaracho Nakagyo-ku, Kyoto 604-8511, Japan TEL: +81-75-823-1482, E-mail: [email protected]

ORCID: Hidenori Takahashi (0000-0001-6887-1724) Yuji Shimabukuro (0000-0003-4090-3995) Daiki Asakawa (0000-0002-9357-8420)

Keywords; Phospholipid, Radical-induced dissociation, Hydrogen atom, Oxygen atom, Hydroxyl radical, Tandem mass spectrometry

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ABSTRACT Gas-phase hydrogen radicals were introduced into a quadrupole ion trap containing singly charged phospholipids to obtain structural fragmentation patterns in tandem mass spectrometry (MS/MS). Saturated and unsaturated phosphatidylcholines were used as a model phospholipid, whose chain-length ranges between 16 and 24.

The MS/MS spectrum yielded a continuous series of

fragment ions with a mass difference of 14 Da, representing the saturated fatty acyl chains.

The

fragment ions corresponding to the double-bond position within a single fatty acyl chain showed a characteristic mass difference of 12 Da.

The detection of these diagnostic product ions enabled the

structural analysis of double-bond isomers of phospholipids.

To further investigate the potential of

radical-induced dissociation for the isomeric analysis of phospholipids, gas-phase hydroxyl radicals and triplet oxygen atoms were employed in tandem mass spectrometry.

The methylene bridges

adjacent to the double-bond positions were selectively dissociated, accompanied by oxidation of the double bonds.

Tandem mass spectrometry incorporating multiple radical species facilitates the

structural analysis of isomeric phospholipids.

Graphic Abstract Sequential Fragments within Acyl-Chains

H• or

Ion Trap MS/MS

OH• O Double-bond Specific Fragments


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INTRODUCTION Tandem mass spectrometry (MS/MS) is an effective analytical technique for the characterization of structurally diverse biopolymers, including proteins/peptides, nucleic acids, carbohydrates, and lipids.

However, distinguishing isomeric compounds using the conventional

low-energy collision-induced dissociation (LE-CID) MS/MS technique often remains ambiguities. This is because LE-CID predominantly cleaves only labile groups of target ions and consequently yields diagnostic fragment ions unsuitable for isomeric structure analysis.

Several radical-induced

dissociation (RID) MS/MS techniques complementary to LE-CID MS/MS have been devised in the last 20 years.

The commercially available RID MS/MS of electron capture dissociation (ECD)1 and

that of electron transfer dissociation (ETD)2 have been successfully applied to identify and characterize proteins and their post-translational modifications (PTMs).3,4

Regarding the

fragmentation process, ECD/ETD are initiated by fragile radical intermediate formation through the electron capture/transfer of a multiply charged peptide.

The radical ions then induce dissociation of

the peptide backbone (N-Cα) bonds, preserving the labile modifications, which enables the isomer analysis of PTM peptides.

However, ECD/ETD cannot be applied to singly charged molecules

because electron attachment to singly-charged molecules produces only neutral species, which cannot be detected by mass spectrometry.

Although peptides and proteins can be ionized into

multiply charged ions by electrospray ionization, small molecules are generally ionized to only singly charged ions.

Therefore, it is quite difficult to characterize small molecules using

ECD/ETD. To address this limitation, the RID technique of hydrogen attachment/abstraction dissociation (HAD) was developed by our research group in 2016.5-7

Since HAD employs

interactions between the target precursor ions and a neutral hydrogen radical (H•) inside an ion trap, even singly charged molecules are dissociated in both positive- and negative-ion modes. HAD is potentially applicable to all types of ionization methods.

Therefore,

Previously, we demonstrated the

usefulness of this technique for peptide sequence analysis combined with matrix-assisted laser



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desorption/ionization (MALDI),8-9 which preferentially generates singly charged molecules. Consequently, HAD was shown to be effective in characterizing not only multiply charged peptides and proteins, but also singly charged small molecules. In this study, HAD was applied to the structural characterization of small molecules of phospholipids for the assignment of double-bond positions in the fatty acyl chains and sn-1/sn-2 positions.

The chain length, degree of saturation, and position of the double bond of the fatty acids

determine their function of biomolecules.

The conventional LE-CID method provides limited

structural information for lipid analysis, because only the labile polar head-group is fragmented by this approach. phospholipids.10

Therefore, LE-CID cannot effectively discriminate the double-bond isomers of In contrast to LE-CID, RID provides detailed structural information for

biomolecules while preserving the labile groups, as reported in our previous study of HAD for PTM analysis5-7 and several studies of RID-based techniques.11-15

To expand the original concept of

HAD-MS/MS, gas-phase hydroxyl radicals (OH•) and triplet oxygen atoms (3O) were also employed for phospholipid dissociation.

Double-bond specific fragmentation within the fatty acyl chains can

be triggered by either OH• or 3O.

Similar double-bond specific fragmentation was reported under

ozone-induced dissociation (OzID) by Thomas and co-workers.16

OzID can identify double-bond

positions and sn- positions, even in complex biological mixtures.17-18

OzID also provides an easily

interpreted fragment spectrum and consequently a higher signal-to-noise ratio.

However, ozone

induces unwanted degradation of the mass spectrometer by oxidizing the electrodes and insulators in the vacuum chamber. Because the life time of ozone is on the order of several minutes,19 ozone interacts with both the target ions and apparatus materials inside the vacuum chamber.

Compared

to OzID, OH• and 3O are readily recombined to molecular form on the chamber wall,20,21 while the accumulated molecules are easily removed through baking the system. Therefore, the unexpected degradation of the mass spectrometer by reactive species is avoided.

Herein, we report how

MS/MS analysis incorporating H•, OH•, and 3O facilitates the structural analysis of isomeric compounds.


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EXPERIMENTS Materials Lipid

standards

(Lyso-PC

18:1(9Z),

PC

18:0/18:1(9Z),

PC

18:1/18:0(9Z),

PC

16:0/24:4(5Z,8Z,11Z,14Z)) and the MALDI matrix were purchased from Avanti Polar Lipids (Alabaster, AL) and Sigma-Aldrich (St. Louis, MO), respectively. dissolved in 100% methanol.

The sample solution was

2,5-Dihydroxybenzoic acid (DHB) matrix was dissolved at a

concentration of 10 mg/mL in 50% acetonitrile/0.1% aqueous tetrafluoroacetic acid (v/v).

The

sample solution (0.5 µL) and matrix solution (0.5 µL) were mixed well on a MALDI sample target. C60 fullerene powder was purchased from Sigma-Aldrich (St. Louis, MO).

C60 powder was

dissolved in 100% toluene, and the solution (1 µL) was spotted thoroughly on the MALDI sample target.

Tandem Mass Spectrometer All MS/MS experiments were performed using a prototype MALDI quadrupole ion trap (QIT) time-of-flight (TOF) mass spectrometer, following our previous study.5

Two

1.5-mm-diameter holes were arranged in the ring electrode of the ion trap for the entrance and exit of radical injection. electrode.

The radical sources were aligned with the axis of the two holes in the ring

A nitrogen laser (wavelength 337 nm) was employed for MALDI ion generation.

The

generated ions were subsequently transported to the ion trap through an aperture on an end-cap electrode.

Helium gas was used for cooling the trapped ions, and argon was used as the collision

gas for conventional CID. isolation (DAWI).22

Precursor ion isolation was achieved by digital asymmetric wave

The reaction time of the radical–ion interaction was set to 0.5–1 s.

The

pressure within the ion trap chamber was maintained below 5 × 10−4 Pa before the radical injection. Trapped ions were extracted with an accelerating voltage of 10 kV in reflectron mode.

Mass

spectra were acquired by averaging 50–100 single shots. H• was generated by passing hydrogen gas through a hot tungsten capillary (length of 50



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mm, inner diameter of 1 mm) heated by thermal radiation from a surrounding hot tungsten filament. The temperature of the capillary was estimated to be approximately 2300 K.

The flow rate of the

input hydrogen gas through the capillary was controlled to below 5 sccm (1 sccm ≈ 4.5 × 1017 atoms/s) by a mass-flow controller (Model 3660, Kofloc, Kyoto, Japan). OH• and

3

O were generated by an in-house built radical source based on a

microwave-driven capacitively coupled plasma generator, as shown in Figure S1.23

A 2.45-GHz

microwave was applied to the quarter-inch (6.35 mm) outer diameter copper tube via an N-type radio frequency (RF) connector and a coaxial cable from a solid-state type microwave power generator (Tokyo Keiki Inc., TMEB101B00B).

The total length of the copper tube was 30.6 cm,

corresponding to 2.5 wavelengths of the 2.45-GHz microwave.

A quartz glass tube (inner diameter:

14 mm, outer diameter: 18 mm) enclosed a tapered copper electrode having a 1-mm-diameter gas outlet fixed to the tip of the copper tube.

The final 15-mm-long section of the quartz glass tube

tapered to the exit hole diameter of 4 mm.

An aluminum cylinder surrounded the tapered quartz

glass tube to maintain a plasma plume at the sharp tapered electrode tip by intensifying the local microwave electric field.

The flow rate of the discharge gas was controlled by a needle valve

(US-916P-P6.35, Fujikin, Japan).

We maintained a background gas pressure of the radical source

and the ion trap at 3 Pa and 0.05 Pa, respectively, which corresponds to a gas flow rate of ≈100 sccm. The higher background pressure (i.e., higher gas flow rate) consequently decreased the product ion intensity due to the decrease of the transport efficiency of the radicals into the ion trap.

The

presence of radical species inside the radical source was confirmed by optical emission spectroscopy acquired with a compact optical spectrometer (Ocean optics USB 2000+ and Ocean optics Flame-S). A Langmuir probe was used to measure the plasma parameters of the electron density and electron temperature.

The optical emission spectroscopy and plasma parameter measurements were

performed in a separate experimental setup which was disconnected from the mass spectrometer as shown in Figure S2.


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Calculations All electron structure calculations were performed with the Gaussian 16 program.24

The

geometries of radicals and molecules were obtained by gradient optimizations with density functional theory calculations using the M06-2X hybrid functional25 and the 6-31G(d) basis set. The structures were characterized by frequency calculations as local energy minima.

To establish

the energetics for fragmentation, the transition state geometries were also optimized at the M06-2X/6-31G(d) level and confirmed by vibrational frequency analysis, showing one imaginary frequency.

The connection of transition states to the reactants and intermediates was checked by

intrinsic reaction coordinate analysis26 of five forward and five reverse steps, starting from the transition state geometry.

The harmonic frequencies obtained by frequency analysis were used to

obtain zero-point energy corrections.



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RESULTS AND DISCUSSION HAD-MS/MS of phospholipid In order to identify the HAD mechanism of phospholipids, we used phosphatidylcholine (PC 18:1(9Z)), which contains a single fatty acid chain, as the model compound.

Figure 1A shows

the HAD spectrum of singly protonated phosphatidylcholine (Lyso-PC 18:1(9Z), 522.36 Da, 10 pmol) formed through the interaction of H• with the isolated precursor ions inside the ion trap.

To

enhance the product ion intensity, supplemental collisional activation27 was applied to the precursor ions after H• injection (HAcaD).

The non-dissociative precursor ions were isolated prior to the

supplemental activation to eject the directly fragmented product ions, which undergo multiple reactions to form unfavorable products for the interpretation of the MS/MS spectrum. activation energy was optimized to obtain sufficient product ions for each analyte. was generated by a thermal cracking cell, following our previous study.5

The

The injected H•

The HAD spectrum of the

model phospholipid shows a continuous series of fragment ions with the mass difference of 14 Da, which represents a CH2 group, between m/z of 312 and 492.

In contrast, the conventional LE-CID

predominantly provides a head-group specific fragmentation, resulting in the undetectable sequential C–C bond cleavage as shown in Figure S3.

Since the HAD spectrum shows the fragmentation

produced by the C–C bond cleavage, HAD-MS/MS is a potentially useful method for the characterization of the double-bond position in fatty acids.

We first focused on the mechanism of

the C–C bond cleavage and then on the analysis of the double-bond position in phospholipids, as highlighted shortly. In order to determine the most probable fragmentation mechanism of HAD, the isotopic distribution of the precursor ion was compared before and after H• injection (shown in the inset of Figure 1A).

To avoid the complexities induced by the overlapping isotopic peaks of the product

ions, only the lightest isotopic peak was isolated prior to the H• injection.

In our previous study of

HAD for peptide analysis, both H• attachments and H• abstractions to and from the precursor ions showed significant results, indicating that H• was preferentially attached to the carbonyl oxygen


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atoms and abstracted from the amide hydrogen or β hydrogen atoms of the peptide backbone. contrast, only H• abstractions are observed after HAD of the phospholipid.

In

This result suggests that

the abundant reactive hydrogen atoms within the fatty acyl chain are dominantly abstracted by injected H•.

Additionally, the fragment ions observed between m/z of 312 and 492 are generated by

the C–C bond cleavage which accompanies double-bond formation.

Therefore, the fragmentation

of phospholipids is probably caused by induced hydrogen abstraction by H•. generated hydrogen-deficient radical underwent a C–C bond cleavage.

Subsequently, the

In this study, we focused on

the formation of the fragment ion at m/z 312, with the mechanism summarized in Scheme 1.

H•

abstracts a hydrogen atom in the acyl chain in Lyso-PC 18:1(9Z) to yield hydrogen-deficient Lyso-PC 18:1(9Z), R1, and H2.

The corresponding transition state barrier for the hydrogen

abstraction (TS1-1) has the energy of 34 kJ/mol; the products of R1 and H2 are more stable than the reactants by 42 kJ/mol.

Because the temperature of H• in the HAD experiment was approximately

2300 K, approximately 50% of H• possesses a kinetic energy exceeding 34 kJ/mol and can react with the phospholipids.

Next, the hydrogen-deficient Lyso-PC 18:1(9Z), R1, undergoes radical-induced

homolytic C–C bond cleavage, leading to double-bond formation. homolytic C–C bond cleavage (TS1-2) is 139 kJ/mol.

The transition state barrier for

The corresponding bond cleavage produced

the intermediate IM1 and the formation of the fragment ion at m/z 312 requires 106 kJ/mol. Because the fragmentation of C–C bond cleavage by HAD proceeds as an endoergic reaction, supplemental activation is necessary to obtain sufficient HAD products in this experiment. Next, we focused on the characterization of the phospholipid by HAD.

Figure 1B shows

the HAD spectrum of singly protonated phosphatidylcholine (PC 18:0/18:1(9Z), 788.6 Da, 10 pmol), which contains two fatty acid chains.

The HAD spectrum of the model phospholipid yields a

continuous series of fragment ions with a mass difference of 14 Da, which represents a CH2 group between m/z 562 and 758.

In addition, fragment ions corresponding to the double-bond position

show the characteristic mass difference of 26 Da between m/z 648 and 674, indicating the HC=CH group.

The fragment ion from the cleavage of the C=C double bond is observed at m/z 660,



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demonstrating that the double-bond position can be identified by HAD. These diagnostic fragment ions enable the discrimination of double-bond isomers of phospholipids.

The inset of Figure 1B

shows the expanded view of the region around the fragment ions at 522 and 524 Da of PC 18:0/18:1(9Z) and that of the regioisomer PC 18:1(9Z)/18:0.

The fragment ions at 524 and 522 Da

represent the product ion from the acyl chain loss at sn-2 for PC 18:0/18:1 and the regioisomer PC 18:1/18:0, respectively.

The fragment ion peaks from the sn-2 chain loss are more intense than

those of the sn-1 chain loss in both cases.

This result suggests that the acyl chain loss from the sn-2

position is preferentially induced by H• abstraction from reactive α-hydrogens of the acyl chain at sn-2.

This feature is effective in the diagnostic process to determine the sn- position of the fatty

acyl chain.


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Figure 1. (A) HAcaD spectrum of Lyso-PC 18:1(9Z) ([M+H]+=522.36 Da). isotopic distributions of precursor ion before and after H• injection. PC 18:0/18:1(9Z) ([M+H]+=788.6 Da).

Inset shows

(B) HAcaD spectrum of

Inset shows detailed product ion spectra between 520

and 530 Da for PC 18:0/18:1(9Z) and for the regioisomer PC 18:1(9Z)/18:0.



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Scheme 1. Fragmentation pathway of PC 18:1(9Z) by H• abstraction. The relative energy (kJ/mol) was obtained by M06-2X/6-31G(d) level calculations, including zero-point vibrational energies.

Generation of gas-phase H•, OH•, and 3O As described above, HAD is initiated by H• abstraction from a phospholipid.

HAD can

cleave the C–C bond in a fatty acyl chain with similar probabilities, because the probabilities of H• abstraction in fatty acyl chain would not depend on the C-C bond position.

As a result, the HAD

MS/MS spectra provided detailed information on the molecular structure of the phospholipids. However, when the target lipids contain complex fatty acid mixtures, the HAD spectrum is


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complicated by the production of many different fragment ions, which impedes the interpretation of the HAD spectrum and deteriorates the signal-to-noise ratio. As described in the introduction, the properties of phospholipids are strongly affected by the double bond position in the fatty acyl chain. reaction with an electrophilic reagent.

The C=C double bond readily undergoes an addition

The reactive species containing oxygen atoms attaches to the

double bond and induces the C=C bond-specific fragmentation.

Herein, we used O2 and H2O as the

radical source, in order to determine the double bond position in the fatty acyl chain.

Firstly, we

analyzed the composition of neutral reactive species generated by the microwave discharge of H2O and that of O2.

The microwave discharge can dissociate a wide variety of reactive gases, yielding

reactive neutral species with lower operating gas pressure (< 10 Pa) and lower input power (< 100 W),28,29 which is suitable for incorporating into a conventional mass spectrometer. Figure 2 shows the typical optical emission spectra for the microwave discharge of H2O with an input power of 100 W and a gas flow rate of 100 sccm.

Under typical experimental

conditions, the estimated electron density and the electron temperature on the capillary axis at 3 cm downstream from the end of the capillary were 1×1010 cm-3 and 3 eV, respectively.

The brightest

peak is observed at 309 nm, corresponding to the ultraviolet-range OH emission band.

Other

intense peaks are found at 656 nm, 486 nm, 434 nm, and 410 nm, corresponding to H-atom emission lines (Balmer-series spectra) and 777 nm and 844 nm for O-atom emission lines.

This result shows

that OH•, H•, and 3O are successfully generated by the microwave discharge of H2O.

Figure S4

shows the optical emission spectrum for the microwave discharge of pure O2 gas, which indicates that 3O is predominantly produced in the discharge process.

In the ultraviolet region of the O2

discharge, the OH emission band does not show sufficient intensity compared to the background molecular spectra.



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Figure 2. Optical emission spectra of the microwave discharge of H2O. (A) Ultraviolet region, (B) visible-light region.

To confirm whether or not the generated radicals are efficiently transported into the ion trap from the radical source, we observed the attachment of H• and O to the fullerene ion C60+•, which is highly reactive with radical species.30

Figure 3 shows the isotopic distribution of C60+• after the

irradiation of the microwave discharge products from (A) H2O and (B) pure O2 at the reaction time of 1 s.

The attachment of more than 4H• and 2O to C60+• are clearly observed, indicating that the

generated radicals are successfully introduced to the ion trap. O attachments are observed.

In the case of pure O2 discharge, only

Assuming that the H• temperature does not alter the H• attachment

coefficient, the number density of H• inside the ion trap is estimated to be 4×1010 cm-3 from the observed H• attachment reaction rate (4 atom attachments per 1 s) and the reported absolute value of the H• attachment coefficient to C60+ (≈1×10-10 cm-3 s).31 Because H• attachment to C60+• is the radical recombination reaction, the threshold energy of the H• attachment to C60+• should be zero or quite low, resulting in the temperature independence of the H• attachment coefficient.

Whereas, the

number density of O and that of OH• cannot be estimated from Figure 3, because we cannot find the absolute value of their attachment coefficients in any literature.

Further study is required to

estimate the number density of radicals inside the ion trap using laser-induced fluorescence (LIF) measurements.32


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Figure 3. Mass spectra of H• and 3O attachments to fullerene ion C60+ induced by (A) H2O discharge and (B) pure O2 discharge at the reaction time of 1 s.

Oxygen Attachment Dissociation of Phospholipid Next, we applied the microwave-driven radical source to the fragmentation of phospholipids. Figure 4A shows the MS/MS spectra of the protonated Lyso-PC 18:1(9Z) obtained by the microwave discharge of H2O vapor.

The irradiation of the microwave discharge products from H2O produced

the fragment ions at m/z 424.21 and 426.22, which were identified as C18H34NO8P and C18H36NO8P, respectively. The proposed structure of the fragment ions is shown in Scheme 2.

The fragment ions

would be formed by oxidation of the C=C double bond accompanied by C–C bond cleavage adjacent to the oxidation site. reference.

To confirm the fragmentation pathway, we used saturated phospholipid as the

As shown in Figure S5, product ions corresponding to the cleavage of fatty acyl chains

were absent from the saturated phospholipid, which suggests that the C–C bond dissociation is triggered by the specific oxidation of the double bond.

The fragmentation of the fatty acyl chain is

induced by the interaction between the C=C double bond and either OH• or 3O, because the



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microwave discharge of the H2O vapor produced H•, OH•, and 3O, as described in the previous section.

Notably, microwave discharge of H2O produced H•, which can induce C–C bond cleavage,

as shown in Figure 1. However, the fragment ions due to the C–C bond cleavage were absent in Figure 4A.

The efficiency of hydrogen abstraction by H• appeared to be lower than that of

oxidation of the C=C double bond by OH• or 3O.

In contrast, the product ions observed below m/z

350 are attributable to H• interaction, because these product ions are also observed in the HAD spectrum of Figure 1. To elucidate the fragmentation process of OH• and 3O individually, we performed an MS/MS experiment on Lyso-PC(18:1(9Z)) using the microwave discharge of pure oxygen gas.

As

shown in Figure 4B, several characteristic fragment ions are observed at m/z 398.19 (C16H32NO8P), 412.22 (C17H34NO8P), 415.21 (C17H37NO8P), 424.21 (C18H34NO8P), and 440.21 (C18H34NO9P). The proposed structure of the fragment ions is shown in Scheme 2. A variety of product ions should be generated by the subsequent reaction following the double-bond oxidation because these product ions are not observed for the saturated phospholipid with no double bonds (see Figure S6). In addition, background oxygen molecules (0.05 Pa ≈ 1.3×1013 cm-3) are more abundant than 3O inside the ion trap because the degree of dissociation of the oxygen molecules is less than several tens of percent in the microwave discharge.

The oxygen molecules are reported to react with alkyl

radicals to yield acetyl peroxide radicals.33

This highly reactive species generates a variety of

fragments.

Therefore, these characteristic fragment ions in the mass spectrum likely arose from

secondary interactions between non-dissociative oxidized precursor ions and the background oxygen gas.

To validate this proposed secondary reaction with background oxygen, oxygen gas was

introduced directly into the ion trap after the injection of the gas-phase H•, OH•, and 3O mixture from the microwave discharge of H2O vapor (Figure 5C). similar to those observed in Figure 5B.

The observed product ions are quite

The secondary interactions between non-dissociative

oxidized ions and background H2O gas are avoided, yielding straightforward double-bond specific fragmentation using the microwave discharge of H2O vapor because the reactivity of H2O vapor is


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quite low compared to that of oxygen.

DFT Calculations for C=C Bond Dissociation As previously described, the double-bond specific fragmentation is initiated by the attachment of 3O and/or OH• to the double bond.

In order to study the most probable fragmentation

pathway, we next investigated the reaction of Lyso-PC(18:1(9Z)) with 3O and OH• by performing DFT calculation. Firstly, we discussed the reaction between 3O and Lyso-PC 18:1(9Z) and the calculated reaction pathway is shown in Scheme 3.

The 3O interacts with the C=C double bond in

Lyso-PC 18:1(9Z) to produce the intermediate complex IM3-1, and the corresponding energy was 22 kJ/mol.

Subsequently, the 3O bonds to the C=C double bond to yield R3 through the transition state

TS3-1.

The transition state barrier for 3O addition is 17 kJ/mol.

R3 is a triplet state diradical,

containing oxyl and alkyl radicals, as shown in Scheme 3. According to the investigation on oxidation of hydrocarbon, the alkyl radical immediately reacts with O2 and we assumed that the alkyl radical in R3 was converted into the peroxide radical.

R3 and O2 adduct on R3 undergo a variety

of radical-induced dissociation, leading to fragment ions at m/z 398.19, 412.22, 415.21, 424.21, and 440.21.

As previously described, the secondary reaction between the analyte radical and O2 was

observed in Figure 5C.

Therefore, fragment ions at m/z 398.19, 412.22, 415.21, 424.21, 426.22 and

440.21 in Figure 5B and Figure 5C are attributable to the interactions between R3 and the abundant background O2 gas in Scheme 3.

The generated radical immediately reacts with O2 and a variety of

fragment ions were generated by subsequent radical-induced dissociation, when O2 was present in the ion trap.

The reaction between the analyte radical and O2 hampers the identification of the

double bond position in the fatty acyl chain. In contrast to O2, H2O does not react with the analyte radical.

As a result, the use of

discharge products from H2O for MS/MS provides the information for identifying the double bond position in the fatty acyl chain.

A comparison of Figure 4A and 4B reveals that the fragment ion at

m/z 426.22 is only generated by H2O discharge containing H• and OH•.

This result suggests that



Page 18 of 28

the fragment ion at 426.22 is attributable to either OH• attachment to [PC(18:1(9Z))+H]+ or H• attachment to R3.

As previously estimated, the number density of H• inside the ion trap is about 3

orders of magnitude lower than that of background O2 or H2O.

In addition, the peak intensity of

[M+H+O]+ is only 3% of the precursor ion intensity in Figure 4A, and the number density of the unstable radical R3 should be much lower than that of the stable oxidized product [M+H+O]+ with an epoxide group.

Thus, it is supposed that the sequential radical reaction of H• attachment to R3 is

a negligible process.

Herein, we focused on the reaction between OH• and the protonated Lyso-PC

18:1(9Z), in order to understand the formation mechanism of fragment ion at m/z 426.22.

Scheme

4 shows the reaction pathway of OH• and the protonated Lyso-PC 18:1(9Z) obtained by DFT calculation.

The OH• interacts with the C=C double bond in Lyso-PC 18:1(9Z) to produce the

intermediate complex IM4-1. was 24 kJ/mol.

The interaction energy of the protonated Lyso-PC 18:1(9Z) and OH•

Subsequently, the OH• bonds to the C=C double bond to yield the radical R4

through the transition state TS4-1.

The transition state barrier for OH• addition is 19 kJ/mol.

As

described in Scheme 1, the transition state barrier for hydrogen abstraction by H• is 34 kJ/mol, which is much higher than that for OH• addition. Consequently, the Lyso-PC 18:1(9Z) preferentially reacts with OH• and the formation of the hydrogen-deficient Lyso-PC 18:1(9Z) radical is suppressed in the presence of OH•.

The radical R4 then undergoes radical-induced C–C bond cleavage,

yielding to enol and alkyl radicals. by 128 kJ/mol.

The corresponding transition state TS4-2 is less stable than R4

Finally, the fragment ion at m/z 426 is formed through the intermediate complex

IM4-2 in exoergic fragmentation at 38 kJ/mol.

The formation process of the fragment ion at m/z

426.22 is reasonably explained by DFT calculation. Regarding the HAD-MS/MS of the phospholipid shown in Figure 1, supplemental activation is necessary to obtain sufficient fragment ion signals.

In contrast, sufficient fragment ions are

obtained without supplemental activation of non-dissociative precursor ions when using the microwave discharge of H2O vapor and that of pure O2 gas.

The irradiation of the phospholipids by

OH• and 3O of the phospholipid yields stable oxidized products and subsequent fragmentations


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proceed simultaneously as exoergic reactions. As a result, supplemental activation is unnecessary. Fragmentation by HAD proceeds via endoergic reactions, which requires supplemental activation to generate fragment ions.

The attachment of 3O and OH• to the double bond induced fragmentation.

In contrast, the triplet state diradical formed by 3O attachment provide more stable singlet state species by intersystem crossing.

Therefore, R3 would result in a stable form of [M+H+O]+ with an

epoxide group, which is more stable than the reactants by 388 kJ/mol (free form of 3O and Lyso-PC 18:1(9Z)), as shown in Scheme 3. radical-driven dissociation.

The epoxide is singlet molecule and does not induce further

In order to investigate the properties of [M+H+O]+, we performed

CID-MS3 experiments for [M+H+O]+ (Figure 5).

The collisional activation for [M+H+O]+ yields

only a head-group fragment at m/z 184.07 with the absence of double-bond fragmentation.

This

result supports the idea that the observed O attached precursor ions are predominantly in the epoxide form which is more stable than the labile head-group.

The CID-MS3 spectrum of [M+H+O]+

generated by H2O discharge also shows an identical result, as indicated in Figure S7. In summary, fragmentation using the microwave discharge of H2O vapor is a potentially useful method for the characterization of the double-bond position in phospholipids.

To confirm

the usefulness of the method, we analyzed PC 20:4/14:0, which contains four double bonds, as a model phospholipid.

Figure 6 shows the fragment ions at m/z 608.39, 648.42, 688.47, and 728.48,

which originate from the cleavage of the C–C bond adjacent to the C=C double bond accompanied by the addition of an oxygen atom.

Therefore, the position of double bonds can be successfully

identified by MS/MS experiments using the microwave discharge of H2O vapor.



Page 20 of 28

Figure 4. MS/MS spectra of phosphatidylcholine (PC 18:1(9Z)) with dissociation induced by (a) microwave discharge of H2O, (b) that of O2, and (c) that of H2O followed by O2 gas injection.


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Scheme 2. Proposed structure of fragment ions observed in Figure 4.



Page 22 of 28

Scheme 3. Fragmentation pathway of PC 18:1(9Z) by 3O attachment. The relative energy (kJ/mol) was obtained by M06-2X/6-31G(d) level calculations, including zero-point vibrational energies.


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Scheme 4. Fragmentation pathway of PC 18:1(9Z) by OH• attachment. The relative energy (kJ/mol) was obtained by M06-2X/6-31G(d) level calculations, including zero-point vibrational energies.



Figure 5.

Page 24 of 28

CID-MS3 spectrum of isolated oxygen attached to phosphatidylcholine (Lyso-PC

18:1(9Z), [M+H+O]+ = 538.34 Da).

Oxygen attachment was induced by the injection of 3O from

the microwave discharge of pure O2 gas.


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Figure 6. MS/MS spectrum of phosphatidylcholine (PC 16:0/24:4) induced by H•, 3O, and OH• mixture (microwave discharge of H2O).



Page 26 of 28

CONCLUSIONS The HAD-MS/MS of phospholipids was demonstrated using MALDI-QIT-TOF MS to identify the double-bond positions within the acyl chains and assign the sn- positions of the acyl chains.

The HAD-MS/MS spectra showed a continuous series of fragment ions with a mass

difference of 14 Da for the saturated acyl chains and characteristic ion peaks with a mass difference of 12 Da for unsaturated acyl chains.

These diagnostic fragment ions enabled the assignment of

double-bond positions within the acyl chains. To expand the original concept of HAD-MS/MS, OH• and 3O were employed for phospholipid dissociation to obtain the double-bond specific fragmentation analogous to that by OzID.

As a result, the methylene bridges adjacent to the double

bonds were selectively fragmented by either OH• or 3O, following the oxidation of the double bonds. These fragment ions are useful for diagnostics to realize detailed structural analyses of lipids in complex biological mixtures using MS/MS.

ACKNOWLEDGMENTS All electron structure calculations were performed at the Research Center for Computational Science, Okazaki, Japan.


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