Improved characterisation approaches for the identification of post-translationally modified peptides by utilising travelling wave-based ion mobility mass
spectrometry
1
1
1
1
1
2
Susan E. Slade; Konstantinos Thalassinos; Gillian R. Hilton; Nisha Patel; James H. Scrivens and Robert H. Bateman
1
Biological Mass Spectrometry and Proteomics, Dept. of Biological Sciences, University of Warwick, Coventry, United Kingdom
2
Waters Corporation, MS Technologies Centre, Manchester, U.K.
Results
The mobilities of all twelve of the doubly charged phosphorylated peptide ions used in this
study were reduced compared to their expected arrival times based on their m/z.
Optimisation of the mobility cell gas pressure and wave height and subsequent analysis of the
mobility data allowed the identification of phosphorylated from non-phosphorylated peptides
especially at higher m/z.
Tandem MS spectra of a doubly charged phosphorylated peptide were obtained by CID in
both the trap and transfer cells. Similar fragmentation was observed in both spectra.
Analysis of the peptides under more stringent denaturing conditions, did not result in a change
in mobility under the experimental conditions used.
INTRODUCTION
Post translational modifications (PTMs) are extremely important and greatly enhance the
diversity and functionality of proteins, can determine the cellular localisation of a protein, its
interaction with other proteins, its turnover, whether it is active or inactive and are implicated in
some disease states. It is estimated that one in three of all proteins are phosphorylated at
some time-point [1]. PTMs can either be permanent or as in the case of phosphorylation
reversible. There are numerous PTMs but the most commonly occurring and most studied are
phosphorylation, glycosylation and ubiquitination. The ability to rapidly identify modified
proteins in complex mixtures would be greatly advantageous.
Ruotolo et al. [2] reported the ability to discriminate between phosphorylated and nonphosphorylated tryptically-generated singly charged peptides using MALDI ion mobility mass
spectrometry based on the change in conformation of peptides that have been modified.
Jensen et al. reported the use of a travelling wave ion mobility device for the separation and
detection of ESI-generated doubly charged modified peptides from an enolase digest. In our
study we further developed the experimental parameters required to optimise the separation
of phosphorylated doubly charged peptides in order to develop a screening process for
modified peptides using the Synapt HDMS system in mobility mode, see Figure 1.
Twelve pairs of peptides identical in sequence were chosen for this study, but one of each pair
had a single phosphorylated residue (serine, threonine or tyrosine) present. In this manner,
we can directly relate the two conformers of control and phosphorylated peptides.
Twelve phosphorylated peptides (Abgent Inc., San Diego, U.S.A.) containing either one
phosphoserine, phosphothreonine or phosphotyrosine residue were selected for this study
and their sequences are shown in Table 1. Each of the peptides and their non-phosphorylated
control peptide was reduced and alkylated with dithiothreitol and iodoacetamide respectively
prior to an overnight in-solution tryptic digest being performed. Thus peptides were
generated that were reflective of those produced during a typical enzymatic digest undertaken
as part of a proteomics study. Similarly, the preparation of the alpha-casein protein was as
described above.
Arrival scan number
68
Our data from the ESI-travelling wave-based ion mobility mass spectrometry experiments
indicates that the mobility plot for the doubly charged non-modified peptide mixtures (m/z 5001200) is approximately linear. Increased arrival times (scan number) were observed for
peptides of increasing m/z, see Figure 2, for all of the experimental conditions used.
63
Alpha-casein tryptic
peptides
Phosphorylated alphacasein tryptic peptides
Linear ( Alpha-casein
tryptic pept ides)
58
53
48
43
38
33
Figure 5. Mass/mobility plot of selected
tryptic peptides from an alpha-casein digest
obtained using a fixed travelling wave height
of 12 V and an IMS cell gas flow of 38
mL/min (phosphopeptides shown in pink).
The requirement to further characterise any identified phosphorylated peptide would require a
tandem MS approach, in order to confirm the affected residue/s A phosphothreonine
containing peptide (IGEG(pT)YGVVYK) was selected for CID performed in either the trap or
transfer cells. The spectra obtained are shown in Figure 9.
28
520
720
920
1120
m/z
55
30
74
Figure 2. Mobility chromatogram of three doubly charged
peptides resolved using travelling wave-based ion mobility
mass spectrometry (from left to right peptides m/z 549.8,
880.5 and 1158.7). The arrival time is noted above each
peak in scan number.
Sequence
Relative
Molecular
Mass
RMM incl.
modifications
[M+2H]2+
CSTRPPTL(pS)PIPHIPR
1850.9227
1907.9442
954.9721
CPGGNIYI(pS)PLKSPYK
1815.8631
1872.8846
937.4423
VSNG(pS)PSLER
1124.4864
1124.4864
563.2432
CLDHEPAV(pS)PLLPR
1625.7637
1682.7852
842.3926
CPLPSGLL(pT)PPQSGKK
1701.8525
1758.8740
880.4370
CNAGSVEQ(pT)PKKPGLR
1763.8390
1820.8605
911.4303
CLMTGDTY(pT)AHAGAK
1618.6521
1675.6736
838.8368
IGEG(pT)YGVVYK
1264.5741
1264.5741
633.2871
CRLMTGDT(pY)TAHAGAK
1774.7532
1831.7747
916.8874
CVYGVSPN(pY)DKWEMER
2054.8268
2111.8483
1056.9242
CGGQYGEV(pY)EGVWK
1653.6535
1710.6750
856.3375
155
IGEGT(pY)GVVYK
1264.5741
1264.5741
633.2871
135
Conventional mass spectrum
Figure 9. CID spectra of a doubly charged phosphothreonine-containing peptide produced in
the trap (upper) or transfer cell (lower) of the Synapt HDMS.
Mobility-selected mass spectrum
Table 1. Table showing sequences and
sites of phosphorylation of peptides used
in this study. Relative molecular mass
prior to and after chemical and enzymatic
cleavage are also shown for each
peptide.
Analysis by means of travelling wave-based ion mobility mass spectrometry
195
115
95
75
55
Mixtures containing a number of phosphorylated and control peptides were prepared in 50%
aqueous acetonitrile containing 0.1% formic acid. Concentrations of the peptides ranged
from 1 - 50 µM in order to produce a spectrum containing similar ion abundances from all of the
peptides present.
The peptides analysed under less denaturing conditions were prepared in 10 mM ammonium
bicarbonate containing 10% methanol.
35
15
520
720
920
1120
m/z
Control peptides using a wave
height of 6 V
Phosphopeptides u sing a w ave
height of 6 V
Control pept ides u sing a w ave
height of 7 V
Phosphopeptides u sing a w ave
height of 7 V
Control pept ides u sing a w ave
height of 8 V
Phosphopeptides u sing a w ave
height of 8 V
Control pept ides u sing a w ave
height of 9 V
Phosphopeptides u sing a w ave
height of 9 V
Control pept ides u sing a w ave
height of 12 V
Phosphopeptides u sing a w ave
height of 12 V
Linear ( Control pept ides u sing a
wave h eight of 6 V )
Linear ( Control pept ides u sing a
wave h eight of 7 V )
Linear ( Control pept ides u sing a
wave h eight of 8 V )
Linear ( Control pept ides u sing a
wave h eight of 9 V )
Linear ( Control pept ides u sing a
wave h eight of 12 V )
Figure 6. Example of a mobility selected region extracted from the DriftScope program (left)
and its effect on the resulting mass spectrum (right).
Figure 3. Mass/mobility plot of phosphorylated
and control peptides obtained using a fixed IMS
cell gas flow of 30 mL/min with variable
travelling wave heights.
Arrival time distribution for phosphopeptide IGEG(pT)YGVVYK
Control peptides using IMS
gas flow 34 mL/min
120
75
Control peptides using IMS gas
flow of 42 mL/min
Phosphopepti des using IMS
gas flow 34 mL/min
110
Phosphopeptides
u sing
I MS
gas
f low
of 42
m
L/min
65
The IMS cell gas was nitrogen, operated at flow rates from 24 - 42 mL/min (4.55 - 7.61 x 10-1
mbar respectively). The travelling wave height was fixed at either 8 or 12 V whilst the IMS gas
cell pressure was optimised. Similarly, during the wave height optimisation the IMS gas cell
flow was fixed at 30 mL/min (5.48 x 10-1 mbar). The travelling wave velocity was fixed for all of
the experiments at 300 m/sec. A scan was acquired every 2 seconds (90 µsec pusher rate)
comprising of 200 mobility bins.
90
Phosphopepti des using IMS
gas flow 30 mL/min
80
Control pepti des using IMS
gas flow 26 mL/min
70
Phosphopepti des using IMS
gas flow 26 mL/min
60
Linear ( Control pepti des
using IMS gas flow 34
mL/min)
50
40
30
520
Linear (Control peptides
using IMS gas flow 30
mL/min)
720
920
m/z
The comparison of the phosphorylated and control peptide mixture under denaturing and less
denaturing conditions was undertaken at an IMS cell flow of 38 mL/min (6.71 x 10-1 mbar) and
a travelling wave height of 12 V. These conditions were also used for the analysis of the alphacasein digest and for the comparison of two peptides of identical amino acid sequence but
differing in their phosphorylated residue.
Control
pept ides
u sing
I MS
gas
flow
of 38
m
L/min
Control pepti des using IMS
gas flow 30 mL/min
100
1120
Linear (Control peptides
using IMS gas flow 26
mL/min)
Two of the peptides used in this study had identical amino acid sequences but the site and
residue affected by the phosphorylation event differed. The mobilities of the two doubly
charged ions having m/z 633.287 were determined and compared to assess if the
phosphorylated residue affected the mobility of the ions through the travelling wave device.
33
Figure 4 (below left) shows the mass/mobility data for the same peptides obtained using a fixed
wave height of 8 V with IMS cell gas flow of 26, 30 and 34 mL/min and shown below right the
data obtained using fixed wave height of 12 V with IMS gas flow of 30, 34, 38 and 42 mL/min.
The samples were infused by means of nanoflow capillaries (Waters Corporation, MS
Technologies, Manchester, U.K.) operated at 1200 V, typically at a flow rate of 100 - 200
nL/min. All analyses were performed on a Synapt HDMS (Waters MS Technologies)
operating in ESI mobility mode (see Figure 1).
The gas used for the trap and transfer cells was argon. The trap gas flow rate was 5 mL/min
(4.25 x 10-2 mbar).
CONCLUSIONS
In order to determine the optimal experimental conditions for the resolution of phosphorylated
from control peptides, the mobilities of the doubly charged peptide ions were determined at a
fixed IMS cell argon flow of 30 mL/min using a travelling wave height of 6, 7, 8, 9 or 12 V. The
mass/mobility plot is shown in Figure 3.
175
A phosphothreonine peptide was selected for a tandem MS experiment undergoing CID in
either the trap or transfer cells. The doubly charged peptide was selected using the
quadrupole then sufficient collision energy was supplied to ensure fragmentation of the entire
peptide in both cells.
Figure 1. Schematic of the Synapt HDMS.
73
Arrival scan number
Methods
Commercially available singly phosphorylated peptides and their non-phosphorylated control
peptides were chemically and proteolytically treated to generate tryptic peptides.
The peptides were analysed by means of ESI-MS and the mobilities of the doubly charged
ions through a travelling wave-based ion mobility device determined. A number of
experimental parameters were optimised and these conditions were used in the analysis of a
tryptic digest of the phosphorylated protein alpha-casein.
Sample Preparation
Arrival scan number
Purpose
To utilise travelling wave-based ion mobility mass spectrometry to facilitate the identification of
potentially post-translationally modified peptides generated during tryptic digestion of a
protein sample.
MATERIALS AND METHODS
RESULTS
Arrival scan number
OVERVIEW
78
34
Control
pept ides
u sing
I MS
gas
flow
of 34
m
L/min
Phosphopeptides
u sing
I MS
gas
f low
of 34
m
L/min
45
Control
pept ides
u sing
I MS
gas
flow
of 30
m
L/min
Phosphopeptides
u sing
I MS
gas
f low
of 30
m
L/min
35
All of the peptides used in this study exhibited reduced arrival time distributions than would be
expected based on the mobility of their control and other non-phosphorylated peptides,
indicating a more compact conformation. All of the phosphorylated peptides used in the study
exhibited similar shifts in mobility indicating that the presence, not the site, of phosphorylation
was a factor in the change in arrival time distribution.
A number of experimental parameters were optimised for the mobility separation of modified
from non-modified doubly charged ions and to date an IMS cell gas flow rate of 38 mL/min
(6.71 x 10-1 mbar) with a fixed wave height of 12 V and a velocity of 300 m/sec appears to be
the most discriminatory, especially at higher m/z. We will be repeating the experiments using
alternative enzymatic/chemical cleavage mechanisms that generate larger peptide fragments
in order to fully utilise the resolving power of the travelling wave device at higher m/z. Further
experimental parameters will also be optimised including travelling wave velocity, ramped
wave heights and velocities and IMS gas.
Arrival time distribution for phosphopeptide IGEGT(pY)GVVYK
Figure 7. Mobility chromatograms of two peptides of identical sequence but differing in the
site and residue of phosphorylation.
Phosphopeptides
u sing
I MS
gas
f low
of 38
m
L/min
55
We have successfully demonstrated the first use of ESI-travelling wave -based ion mobility
MS to distinguish a number of singly phosphorylated, doubly charged tryptic peptide ions from
their non-modified control.
A sample containing both phosphorylated and control peptides was prepared in both
denaturing and a more physiological buffer. Mobility data were obtained for a number of
peptides, two of which are shown in Figure 8.
It was possible to identify a number of potentially modified peptides from an alpha-casein
digest due to their differing mobility through the T-wave device, using the experimental
conditions described above. The use of the DriftScope data processing tool allows sections of
the mass/mobility plot to be extracted from the raw data allowing more rapid identification of a
peptide due to its charge state. With further optimisation of the experimental conditions, it is
envisaged that an extracted spectrum from the raw ESI-TWIM MS data, can be generated
using DriftScope that is optimised for the identification of phosphorylated peptides.
The mobilities of the doubly charged tryptic peptides were unaffected (within the
reproducibility of the experiment) by the denaturing capability of the solvent system used.
Linear
( Control
pept ides
u sing
IMS
gas
f
low
of 42
m
L/min)
Linear
( Control
pept ides
u sing
IMS
gas
f
low
of 38
m
L/min)
25
33
57
33
58
Linear
( Control
pept ides
u sing
IMS
gas
f
low
of 34
m
L/min)
15
520
720
920
1120
Linear
( Control
pept ides
u sing
IMS
gas
f
low
of 30
m
L/min)
m/z
We were unable to distinguish between two peptides of identical sequence that differed in the
site of phosphorylation. We envisage that a combination of experimental and molecular
modelling experiments will be required to establish the extent of conformational change on
phosphorylation in the peptides studied.
Figure 4. Mass/mobility plots of phosphorylated and control peptides obtained using fixed
travelling wave heights of 8 V (left) and 12 V (right) with IMS cell gas flow indicated.
We will be converting the arrival time distributions into rotationally averaged cross sections for
each peptide based on a calibration created using conventional drift tube mobility/cross
section measurements on a further set of tryptic peptides.
Based on the results obtained, all further mobility experiments were performed using an IMS
gas flow of 38 mL/min and a travelling wave height of 12 V.
CID experiments can be conducted in both the trap or transfer cells of the Synapt HDMS
system both resulting in fragmentation, predominantly a y-ion series that can be interpreted
from the N– to C- terminus and includes the loss of a phosphothreonine residue.
A tryptic digest of the phosphorylated protein alpha-casein was performed and the peptides
analysed as described above. The mobility data of a number of non-phosphorylated peptides
was obtained and plotted on a mass mobility graph, see Figure 5. The mobilities of a number
of known phosphorylated alpha-casein peptides were obtained, aided by the use of the
mobility-specific data processing tool in DriftScope (Waters MS Technologies), see Figure 6.
Figure 8. Mobility chromatograms of two phosphorylated doubly charged peptide ions (m/z
633.3 and 956.0) obtained using a fixed travelling wave height of 12 V and an IMS cell flow of
38 mL/min under denaturing (left) and near-native (right) conditions. The arrival time is noted
above each peak in scan number.
REFERENCES
[1]. Kumar, R., A.E. Gururaj, and C.J. Barnes, (2006) Nat. Rev. Cancer 6: 459-71.
[2]. Ruotolo, B. T., Verbeck IV, G. F. Thomson, L. M., Woods, A. S., Gillig, K. J. And Russell, D. H. (2002) Journal of Proteome Res. : 303-06.
[3]. Jensen, O. N., Larsen, M. R., Wildgoose, J. Bateman, R. H., Giles, K., Pringle, S., Hughes, C. And Langridge, J. Poster presentation ASMS 2006.