J. Mass Spectrom. Soc. Jpn.

Vol. 55, No. 5, 2007

Tandem Mass Spectrometry Using an Axially Resonant
Excitation Linear Ion Trap
Yuichiro H6H=>BDID, Hideki H6H:<6L6, Masuyuki SJ<>N6B6,
Hiroyuki S6I6@:, Takashi B676, and Izumi W6@>
Central Research Laboratory, Hitachi, Ltd., Kokubunji, TOKYO, JAPANWe performed collision-induced dissociation (CID) and electron capture dissociation (ECD) analyses using a
new ion trap: an axially resonant excitation linear ion trap (AREX LIT). Unlike a conventional linear ion trap,
the AREX LIT can trap and detect low m/z fragment ions, such as immonium ions and iTRAQTM reporter ions,that are produced by CID. This capability to detect low m/z fragments is realized by CID excitation in a nearly
harmonic direct current potential along the linear axis. In contrast, CID excitation in a conventional linear ion
trap is in a pseudo-harmonic radio frequency potential that destabilizes fragment ions whose m/z is less than 1/4
that of the precursor ions. ECD was achieved in an axial magnetic ﬁeld of about 0.2 Tesla superimposed along
the AREX LIT. We observed that sequential ECD/CID inside the trap improved the sequence coverage of a
peptide.
ῌReceived May 30, 2007; Accepted June 27, 2007῍

1.

Introduction

Tandem mass spectrometry (MS/MS) using a twodimensional quadrupole ion trap (or linear ion trap,
LIT) is a powerful tool for analyzing proteins,1) peptides,2) carbohydrates,3) metabolites,4) and other compounds. LITs are widely used as mass selective ejection devices in low-resolution mass spectrometers5)7) or
as fragmentation and precursor selection devices inhigher-resolution hybrid mass spectrometers.8)10)
However, the conventional LIT has di$culties as a
fragmentation device; it has low m/z fragment loss andan isolation shift caused by magnetic ﬁeld perturbations as described below.
Collision-induced dissociation (CID) is the mostcommon dissociation method for MS/MS analysis of
LITs,2)6), 8), 9) because it is inexpensive and has a simple
conﬁguration. However, the CID inside the conventional LIT that is performed by resonant excitation cannot
detect low m/z ions, because the detectable mass range
and the dissociation e$ciency are in a “trade-o#,” asdescribed below. E#ective dissociation requires a highpotential where ions are oscillated and activated bycollisions with the bath gas.11) The minimum m/z ofproduct ions, Mmin, that can be stabilized inside quadrupole ion trap (QIT), which includes LIT, is
Mmin῏

q
Mp ,
qej

(1)

where Mp is the m/z of the precursor ion, q is thestability parameter for the precursor ion, and qej is aconstant value of 0.908. Because the oscillation poten Correspondence to: Yuichiro H6H=>BDID, Hitachi, Ltd.,
Central Research Laboratory, Higashi-koigakubo 1280,
Kokubunji-shi, Tokyo 1858601, JAPAN, e-mail: yuichiro.
hashimoto.ur@hitachi.com

tial of a QIT, which corresponds to the pseudo potential
formed by the trapping radio frequency (RF) ﬁeld, is
proportional to the square of the stability parameter q
for the precursor ion, dissociation usually requires ahigh q of over 0.200.25.9) For q῏0.23, Eq. (1) becomesMmin῏0.25Mp .

(2)

Therefore, product ions, whose m/z is less than about
1/4 that of the parent ion, are neither trapped nor
detected. To overcome this problem, two CID methods,
high amplitude short time excitation (HASTE)12) andpulsed q collision-induced dissociation (PQD),13) werereported. In HASTE, after precursor ions are excited at
a high q of 0.25 for a short duration of 12 ms, q isdecreased to 0.10 by decreasing the trapping RF voltage over a short period of 100 ms. Low m/z productions, which are produced after the q decrease, can betrapped inside the trap. PQD obtains a relatively highdissociation e$ciency of over 60ῐ for some ion species.
However, because the optimum excitation
strength and duration are dependent on the lifetimes of
the ion species after activation, low dissociation
e$ciencies of less than 20ῐ have been also reported
for some species.13)
Electron capture dissociation (ECD) in an LIT was
also achieved by perturbation of an axial magnetic
ﬁeld of 50200 mTesla.14)16) The axial magnetic ﬁeld
a#ects the ion’s motion as,F῏ev῎B(3)

where the magnetic ﬁeld

0

B
0

Bz(x, y, z)

is variable in space. Because the precursor ion selection and CID of the conventional LIT increase the 339 

Y. Hashimoto et al.

radial velocity (x, y-directions), axial magnetic ﬁeld willcause mass shifts. We estimated that the mass shift
caused by an axial magnetic ﬁeld of 200 mTesla is 10
Th for an m/z of 1,000 at q῏0.10.16) Because themagnetic ﬁeld made by a cylindrical magnet is not
uniform inside the LIT; mass shifts may vary in space,which is not favorable for mass spectrometric performance.
Our previously reported axially resonant excitation
linear ion trap (AREX LIT) is capable of high e$ciency
mass selective ejection (over 60ῑ) from an LIT.7) The
LIT has a set of electrodes between the quadrupole
rods that make the direct current (DC) potential approximately harmonic along the central z-axis. Inside
the DC quasi-harmonic potential, ions with a speciﬁc
m/z range can be resonantly oscillated and ejected in
the axial direction by applying a supplemental
alternating current (AC) ﬁeld. In this paper, we reporton solving the above two problems a#ecting CID and
ECD in RF ion traps, by using this new trap.
2.

Experimental

Samples were ionized using a gas-assisted electrospray ion source (ESI source) at a sample ﬂow rate of
5 mL/min. Ions were introduced from the ESI source
through high-pressure multipole rods into a ﬁrst linear
ion trap (hereafter pre-LIT). Ions that were massselected in the pre-trap are put into the AREX LIT.9)
The details of the AREX LIT experimental setup (Fig.
1) were described in previous reports.7) The di#erences
between the devices of the previous reports and thisone are the addition of a cylindrical magnet and anelectron source in front of the inlet lens. The vanepotential (D0), which is a DC potential of the vane
electrodes relative to the quadrupole rods’ DC o#set,
makes a potential approximately harmonic along thecentral axis of the quadrupole ﬁeld. A supplemental
AC voltage (ῐ῍20.0 V (0-peak), 5῍100 kHz) applied
between the front and rear vanes oscillates ions with a
speciﬁc m/z in the axial direction, which enables precursor ion selection and CID. The resonant frequency
in the axial direction, f, is
f῏

1
2p

D
῎ 2neA
mL

2 0
2

(4)

Fig. 1.

Structure of AREX LIT with an ECD magnet.

vane length. The cylindrical magnet forms a magnetic
ﬁeld of about 200 mTesla along the quadrupole axis.
The electron source in front of the inlet lens introduces
electrons with kinetic energies of 0῍3 electron volts;
the kinetic energy and the electron current are controlled by the potential di#erence between the electron
source and the linear trap o#set.
The MS/MS analysis measurement sequence (ECD
followed by precursor selection and CID) is depicted inFig. 2. ECD and precursor ion selection processes areomitted for CID. The measurement sequence consistsof accumulation, ECD, precursor ion selection, CID, andejection. During the accumulation period, ions thatwere mass-selected in the pre-trap are put into the
AREX LIT, where the vane electrodes make an axialtrapping ﬁeld and quadrupole rods make a radialtrapping ﬁeld. During the ECD period, the vane potential (D0) of 1῍5 V is applied, so that ions are weakly
focused near a central point O. During the precursor
ion selection and CID periods, the vane potential rises
to 20῍40 V, and the supplemental AC voltage is applied. Oscillated ions are ejected or collisionally activated with mass selectivity. Helium is used as a 5
mTorr bath gas in the AREX LIT; the base pressure of
the chamber is about 3῎10ῌ5 Torr. After the MS/MS
analysis, ions are detected by using an orthogonal acceleration time-of-ﬂight mass spectrometer (oaTOFMS)
with a mass resolving power of over 5,000.

where e is the elementary charge, A2 is a coe$cient of
0.833, D0 is vane DC potential, m is ion mass, and L isFig. 2.

Measurement sequence for ECD followed by CID.
ῌ 340 ῌ

Tandem Mass Spectrometry Using an Axially Resonant Excitation Linear Ion Trap

3.

Results and Discussion

3.1 CID experiment
Figure 3 shows the CID e$ciencies of AREX LIT andconventional LIT versus the stability parameter q. CID
e$ciency is deﬁned by,
ECIDῌῌFragment
Precursor.

(5)

where Precursor means the intensity of precursor ions
before CID and ῌFragment means the sum of theintensities of fragment ions after CID. Because thedetection e$ciency of lower m/z range is higher thanthat of higher m/z range in the oaTOFMS, CID e$ciency became over 1.0. As the basis for comparison in theexperiment, we used a conventional LIT/oaTOFMS
mass spectrometer, whose conﬁguration and measure-Fig. 3.

CID e$ciencies versus q for AREX LIT (῎) anda conventional LIT (῏). Sample: reserpine (m/z609.3), D0: 40 V, CID period: 5 ms.

Fig. 4.

CID mass spectra by AREX LIT (A) and conventional LIT (B). Sample: iTRAQTM-LCTVATLR,
m/z 533.8, D0: 40 V, CID period: 5 ms, qῌ0.10(AREX LIT), qῌ0.23 (conventional CID).

ment sequence were described previously.9) This result
supports the conclusion that dissociation of the conventional LIT only proceeded with a high q of over
0.20, because the oscillation potential was not su$cientfor dissociation with a lower q. As described in the
introduction, the fragment ions whose m/z is less thanabout 1/4 that of the precursor ions will be neither
trapped nor detected by the conventional LIT. On theother hand, e#ective dissociation occurred with low q
values of less than 0.10 in the AREX LIT, because theoscillation potential formed by the vane DC potential
did not a#ect the trap’s stability.
Figure 4 shows CID results for a peptide that islabeled with iTRAQTM reagent.17) In the AREX LIT,
low m/z fragment ions such as iTRAQTM reporter ions(m/z 114.1 and m/z 117.1) and iTRAQTM intact ions(m/z 145.1) were detected as strong peaks in the spectra. This CID spectrum is similar with the other CID
spectra of iTRAQTM-labeled sample that were obtainedby quadrupole time-of-ﬂight mass spectrometer(QqTOFMS) or tandem time-of-ﬂight mass spectrometer (TOF/TOF).18) On the other hand, we chose qῌ0.23
for the e#ective CID in the conventional LIT experiment, because CID e$ciency decreases with lower q, as
shown in Fig. 3. Equation (2) indicates that no fragment peaks with m/z of less than 130 would be observed. No iTRAQTM reporter ions (m/z 114.1 and m/z
117.1) were observed in Fig. 4(B), which is consistent
with the above discussion.
3.2 Sequential ECD/CID
Figure 5 shows the mass spectra gotten by ECD (A)and sequential ECD/CID (B). The ECD target wasFig. 5.

ῌ 341 ῌMass spectra by ECD (A) and sequential ECD/
CID (B). Sample: neurotensin, m/z 553.8, ECD
period: 15 ms, CID period: 5 ms.

Y. Hashimoto et al.

triply-charged ions of [M῎3H]3῎. Dissociation sites
identiﬁed by ECD and sequential ECD/CID are
depicted in the upper right. Not only fragment ions but
also charge-reduced radical ions of [M῎3H]2῎ῌ wereobserved in the ECD spectra. After ECD, we selected
the charge-reduced radical ions of [M῎3H]2῎ῌ as the
precursor ions of the following CID. By axial resonant
excitation, the charge-reduced ions were collisionallydissociated into c, z fragment ions that are characteristic of ECD/CID fragments. Sequential ECD/CID improved the sequence coverage of the peptide comparedwith ECD alone (bold-line cleavages in Fig. 5(B) aredissociation sites only observed by sequential ECD/
CID). In the AREX LIT, ions can be oscillated massselectively by a resonant excitation within the axial DC
harmonic potential. The ion motions during isolation
and CID periods are little a#ected by the magnetic ﬁeld
(Eq. (3)), because both the direction of the magnetic
ﬁeld and the ion velocity are axial.
4.

3)

4)
5)
6)
7)
8)

9)
10)
11)
12)

Conclusion

We performed ECD/CID in a linear ion trap, called anaxially resonant excitation linear ion trap (AREX LIT),
with an axial magnetic ﬁeld. AREX LIT can detect low
m/z fragment ions that cannot be mass analyzed withconventional linear ion traps. Precursor ion selection
and CID can be achieved by axially resonant excitation
because the ion’s axial motions are not a#ected by theaxial magnetic ﬁeld. These superior properties of the
AREX LIT will help to improve proteome analyses.

13)

14)
15)

Acknowledgments

16)

We would like to thank Mr. H. Toyama and Mr. T.
Iwata of our laboratory for providing the electricalsupply system for this instrument. We also gratefully
acknowledge ﬁnancial supports from Hitachi Hightechnologies Corporation.

17)

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18)

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Keywords: Axially-resonant excitation linear ion trap
(AREX LIT), Sequential ECD/CID, Low m/z fragment detection

῍ 342 ῍