ETD-IMS and LC-ETD Studies on a QTOF Mass Spectrometer
Jeffery Brown, Steven Pringle, Keith Richardson, James Langridge, Richard Chapman, Iain Campuzano and John Hoyes
Waters Corporation, Wythenshawe, Manchester, UK.
OVERVIEW
•
•
•
A hybrid Q-IMS-TOF (Waters Synapt)
has been modified to enable Electron
Transfer Dissociation (ETD) on LC time
scales.
A new glow discharge reagent ion
source is described.
Ion mobility measurements of ETD
product ions are also presented.
Applied voltage –500V
Isolation
Valve
Discharge
Pin
Analyte
Sampling
Cone
Extraction
Vapour Inlet
Tube
Vial of Reagent
Crystals i.e.
Azobenzene or
Fluoranthene
INTRODUCTION
ETD (Electron Transfer Dissociation) is a MS/MS
technique in which precursor cations are reacted with
radical reagent anions. Electrons transfer from anion
to cation promoting fast and randomised dissociation
compared to collision induced dissociation (CID). ETD
product ion spectra contain predominantly "c" and "z"
type ions and post translational modifications often
remain
intact
providing
valuable
sequence
information.
Bovine serum albumin (BSA) tryptic peptides (250fm
and 50fm) were separated by nanoAcquity UPLC and
a selection of triply charged precursor mass
chromatograms are shown in Figure 4. Figures 5 to
9 show the LC-ETD spectra annotated and database
matched to BSA with high probability (Mascot scores
inset). Similar high quality data was observed for the
50fm injections (see Figures 10
and 11 for
comparative data). The anion source was activated
between 10% and 20% of the analysis time using
Waters Research Enabled Software “WRENS”. Data
were acquired at 1 spectra/second.
ETD
m/z 450
3+
2+
IMS
CID (40eV)
m/z 450
3+
TOF
TOF
1+
Figure 8: ETD m/z 381.57 BSA KQTALVELLK
2+
Figure 2: Cross section of modified z-spray ion source block
incorporating glow discharge pin
3+
1+
Trap
Twave
IMS
Twave
Precursor (M+3H) 3+
Transfer
Twave
Figure 5:ETD m/z 488.53 BSA TCVADESHAGCEK+2 CAM (C)
Figure 12: m/z versus drift time for the ETD products of
Substance-P m/z 450 with 1eV collision energy into Transfer guide
Figure 14: m/z versus drift time for ETD products of Substance-P
m/z 450 with 40eV collision energy into Transfer guide
A
A
Figure 9: ETD m/z 580.94 MPCTEDYLSLILNR+CAM+Metox
(a)
3+
3+
2+
1+
2+
B
B
Figure 3: Potential energy schematic depicting flow of anions
and cations into Trap ion guide
C
(b)
RESULTS
1+
Figure 13: A multiply charged product ions and B singly
charged product ions extracted of data in Figure 11
m/z 488.53
Figure 6: ETD m/z 435.90 BSA HLVDEPQNLIK
Figure 10: ETD m/z 435.90 (a) 250fm and (b) 50fm injected
m/z 435.90
(a)
3+
m/z 547.31
3+
2+
2+
•
•
m/z 381.57
•
•
•
m/z 580.94
Figure 1: Synapt — ETD is performed in the TRAP
travelling wave ion guide
ETD
3+
METHODS
ETD was performed on a hybrid Q-IMS-TOF (Waters
Synapt) (Figure 1), a high voltage discharge pin was
added to the ion source to generate reagent anions
(Figure 2). Analyte cations were generated using the
standard nanospray source via infusion or UPLC
system. For ETD, the ion source polarity and the
quadrupole set mass were sequentially switched to
deliver anions and cations into the TRAP travelling
wave (TWAVE) ion guide where they reacted to form
ETD product ions (Figure 3). Product ions were
optionally separated by ion mobility in the IMS TWAVE
ion guide or were accelerated into the TRANSFER
TWAVE ion guide to cause 2nd generation collision
induced dissociation (CID) ions prior to mass analysis
in the TOF.
IMS
nanoAcquity
75um x 100mm
BEH column
1uL/min
BSA digest 50fm
30 min linear
gradient
1+
1+
(b)
Figure 4: LC mass chromatograms of ETD precursors
Figure 7: ETD m/z 547.31 BSA KVPQVSTPTLVEVSR
Figure 11: ETD m/z 381.57 (a) 250fm and (b) 50fm injected
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Figure 12 depicts m/z versus drift time for the ETD
products of Substance-P m/z 450 (infusion of 1pm/ul)
Multiply and singly charged product ions were
extracted from the data in Figure 12 and are shown
in Figures 13 A and B.
For ETD-IMS-CID (Figure 14) , the collision energy
between the exit of the IMS cell and the entry of the
Transfer cell was increased from 1eV to 40eV. This
resulted in vertical bands of second generation
product ion data. Data from bands A, B and C were
extracted, annotated and displayed in Figure 15. For
example, band C shows the second generation
fragments of the single charge reduced ETD product
ion at m/z 1347.7, these ions are predominantly “c”
type ETD process ions despite being activated by CID
This suggests that these products may have been
held together non-covalently prior to the CID
activation.
Figure 15: Annotated product ion spectra extracted from the
vertical bands A,B and C of data in figure 14
CONCLUSION
•
•
•
LC-ETD has been demonstrated on a hybrid
Q-IMS-TOF instrument (Synapt).
A new glow discharge ionisation source
provides an effective reagent source for ETD.
Ion mobility enables novel experiments such
as ETD-IMS and ETD-IMS-CID.
©2009 Waters Corporation v1