Protein Phosphorylation Sites Determination Using A Microfluidic Chip Interfaced With ETD Ion Trap And Q-TOF Mass Spectrometry
MSB-2007
P03-59-M
Ning Tang, Patrick Perkins, Christine Miller, & Tom van de Goor; Agilent Technologies, 5301 Stevens Creek Blvd., Santa Clara, CA 95052, USA
Experimental
Introduction
Post-translational modifications (PTM) of proteins, such as
protein phosphorylation, play important roles in regulation
of many cellular functions. The precise determination of
the location of phosphorylation is crucial to the
understanding of the regulation mechanism.
Many
biological and analytical methods have been developed to
study protein phosphorylation, however, most of them
failed to pinpoint the exact location of phosphorylation if
multiple phosphorylation sites are present. Electron
transfer dissociation (ETD) is a new technique analogous
to electron capture dissociation (ECD) where an electron is
transferred from a radical anion to a multiply-protonated
peptide ion, inducing c and z ions indicative of the peptide
sequence.
An additional observation for the ETD
technique is that the phosphate remains attached to the
amino acid during the collision process and allows the
specific location of the phosphate group to be observed.
Results and Discussion
LC/MS/MS
Data were collected on a HPLC-Chip/MS interfaced to
either a 3-D ion trap equipped with ETD (Agilent 6340 Ion
Trap LC/MS), or Agilent 6510 QTOF. HPLC-Chip is a
microfluidic chip-based device which integrates sample
preparation (trapping column), analytical separation
(nanocolumn) and nanoelectrospray formation (emitter tip).
A multi-step gradient from 3% to 90% acetonitrile (+ 0.1%
formic acid) was used for the chromatography with a total
run time of 45 minutes. The flow rate was set at 600
nL/min.
Data processing was done using Spectrum Mill MS
proteomics workbench software. The spectra were
searched against the SwissProt database using
carbamidomethylation as the Cys modification.
Spectrum Mill can search CID and ETD mixed
LC/MS data.
Comparing QTOF CID, IT CID and IT ETD for
phosphopeptides
x10 4
QTOF CID MS/MS of TRDyETDYYRK
Comparing QTOF CID, IT CID and IT ETD for
phosphopeptides
x10 3
1.8
516.7865
6.5
2.8
V a1
72.0810
541.6345
0
P,R
0.4
0.2
216.0413
Y
100
y42+
b4
b3
315.1744
E b1
b2
150
200
250
300
350
400
PI-28
1.6
183.1475
y5
b6
744.3650
y6
800
974.4656
Preliminary studies were performed on synthetic
phosphopeptides to demonstrate the phosphoserine/threonine remained intact in the ETD fragmentation
process. It was also observed that ETD produced more
fragments on the triply charged precursors than the doubly
charged precursors. Further studies were performed on a
proteolytic digest of mitogen-activated protein (MAP)
kinase using LC/MS/MS. A targeted method was set up
using CID neutral loss to monitor peptide phosphorylation
with a subsequent ETD spectrum of the same precursor
ion. The results showed improved protein identification
scores combining both QTOF and ETD IT data while
providing site determination of protein phosphorylation.
Experimental
Materials
Phosphopeptide standard mixture was purchased from
Invitrogen (Eugene, OR). Activated mitogen-activated
kinase (MAP kinase/ Erk1) was purchased from Upstate
Cell Signaling Solutions (Temecula, CA). All proteins were
digested with trypsin using a protocol based on 2,2,2trifluoroethanol as the denaturant. The digests were
aliquoted, dried and stored frozen until use. HPLC grade
solvents were obtained from Burdick & Jackson.
I
0.8
762.4242
y112+
459.6012
PI
119.0479
600
700
800
900
1000 1100 1200 1300 1400
Abundance vs. Mass-to-Charge (m/z)
850
900
950
150
200
1800
1900
2000
2100
2200
250
300
350
650
700
750
800
850
900
y4++
x105
315.1
b8
0.6
Ion Trap CID MS/MS of VPIPGRFDRRVtVE
[y18-H3PO4]2+
2+
y161071.0
2
y4
b5
466.2 b
4
486.2
476.4
b8
729.4
[M+3H-H3PO4]3+
[b11-17]2+
[y10-H3PO4]2+ b7
y11-H3PO4
b12 y11b13
b10-17 y
9
625.5
400
600
800
1000
1200
1400
400
ETD
1600
1800
m/z
0.0
400
600
800
1000
1200
1400
1600
m/z
706.3
Ion Trap ETD MS/MS of ELEELNVPGEIVEsLsssEESITR
b8
882.5
0.0
400
600
800
1000
1200
1400
1600
m/z
TR
Ion Trap ETD MS/MS of VPIPGRFDRRVtVE
R
VPIPG
t
EV
F
D
R
VR
c5
R
D
t
RV
R
F
KR
PV
z2
z3
410.3
637.5
570.4
z
5
z6
z7
1055.6
I
y
D
K
400
600
800
1000
1200
1400
L
[M+H]+
T
c5
c4
614.4
I
S
0.4
z4
z2
0.2 260.1
z3
s
s E E SI T
RT
851.8
1703.7
z4
s
E
s
[M+2H]2+
746.3
z5
c7
c6
976.4
875.4
c8
z10
z9
1314.5 1430.3
z8
1091.2 1202.5
c10
z3
460.46
z5
589.5
373.3
[M+4H]4+
781.8
z6
718.6
C192+C
C172+
z7
C152+ C162+
2+
C
2+
1203.7 20 21
1268.8
C182+
C242+
1484.4
C 2+
C222+23
200
400
600
800
1000
1200
1400
z18
1600
1800
2000
m/z
c9
z7
0
c13
200
400
600
800
1000
1200
1400
1600
m/z
z12
m/z
Figure 2. QTOF CID, Ion trap CID and ETD spectra of
the phosphothreonine peptide VPIPGRFDRRVtVE.
QTOF CID produces low mass MS/MS fragments
including immonium ions. The dominant fragment
in ion trap CID spectrum ins the loss of phosphate.
Ion trap ETD provides the information of the exact
location of the phosphorylation.
c9++
c13++
400
600
1400
L
GT
t
1600
E
HD
1800
y
H
2000
V
E
m/z
ATR
PDAI
[M+2H]2+
1168.0
z6
802.3
z
1200
766.3
4 517.2 c
c6
5
z2 z3 430.3 543.2 680.4
c4
261.16 414.1
[M+3H]3+
331.2
200
1000
T G F
tLF
c13
c10
c14++
830.8
1090.5
c8
c9
1033.7
c11
z9 c12
1243
932.5
800
1000
1532.6
1237
1200
z11
1351
1402
1400
z15
c14
1922 c
16
z13 1661 z14 c
15
1654
1600
1790 1904
1800
2002.9
2000
m/z
Figure 5.
Comparison of CID and ETD of
phosphopeptide IADPEHDHTGFLtEyVATR. The most
prominent peak in CID corresponds to the loss of
phosphate group. ETD produces nearly complete
sequence coverage.
Conclusions
0.0
c11
1573.7
1492.8 1591.1
1600
1563.0
0.8
0
200
[M+2H]2+
1.0
R
275.1
z2
c12
c11
1311.7
Y
c2
z9
c9 z8
TE
Y
390.1
503.3
1241.7
c6
[M+3H]3+
D
568.5
2000
c7
230.1
D
T
1042.2
0.6
4000
[M+2H]2+
2000
Y
E
[M+3H]3+
1722.1
861.4 c
8
899.4
784.5
y
c3
[M+H]+
4000
Y
E
481.3
6000
I
6000
V
GPI
D
[M+3H]3+
x10 5
Ion Trap ETD MS/MS of TRDyETDYYRK
8000
E
z5
673.2
3
0
1.0
y11-H3PO4++
657.3 y11++
800
DH
z
200
200
371.2
822.3 894.8
600
600
y12-18 y13 y16-H3PO4
0
400
IADP E H
RT A V y
0.5
1.5
y17-H3PO43+
b15++
684.0
y9++
b ++ 943.8
b12++ 14 b15-H3PO4++
y4
347.4 446.5
b6
1074.4
541.8
200
y3
200
[M+3H-H3PO4]3+
y6-H3PO4++
CID
0.0
y172+
858.3
b3 423.1
y3
Figure 1. Diagram of the ETD ion trap mass spectrometer.
The ETD reactant was generated by a small negative
chemical ionization source (NCI). Fluoranthene was used
as the reactant. The fluoranthene was sublimed and
combined with methane gas in the presence of electrons
emitted from a filament. The electrons were captured by
the fluoranthene molecules to make fluoranthene radical
anions. Analyte cations were first accumulated in the ion
trap and the selected precursor was isolated. During this
process, the flow of the reactant radical anions was closed
by a voltage gating process. Alternatively, the negative
ions were then pulsed into the ion trap. Electron transfer
and fragmentation occurred in the ion trap and the
fragments were scanned.
0.8
0.4
983.8
y4
629.3
y6++
373.1
1.0
0.5
[M+3H-H3PO4]3+
559.5
Ion Trap CID MS/MS of TRDyETDYYRK
b8
882.4878
400
450
500
550
600
Abundance vs. Mass-to-Charge (m/z)
~x4
x104
1.0
6
y132+
1.5
x10 6
1700
Ion Trap CID MS/MS of ELEELNVPGEIVEsLsssEESITR
0
100
1600
1000 1050 1100 115
4
50
1500
The extracellular signal-regulated kinase (ERK)
pathway is essential to transmitting signals from
many extracellular agents to regulate cellular
process. The signaling via the ERK cascade is
mediated by sequential phosphorylation and
activation of protein kinases within the cascade
pathway. The active Erk1 kinase was digested and
analyzed by alternating CID and ETD on ton trap in a
single LCMS run.
0.2
811.4148
371.2205
247.1278
0.2
Positive
Ion
Packet
500
y122+
GRFD-NH3
a2
0.4
Negative
Ion
Packet
400
657.3565
b2
0.6
Ion
Trap
With
Ions
300
x10 6
1
ESI
Positive
Peptide
Ions from
LC Chip
200
[M+2H-H3PO4]2+
b14 b -H PO
1607.8407
16
3 y4 -H PO
1789.9330 1944.8196
16
3
4
2140.7659
y16
1074.4179
y102+
1.2
++
b3
b11
1266.6345
b7
1.4
+
b
b5770.40636
y6
b8
y7
858.3370
b5
450 500 550 600 650 700 750
Abundance vs. Mass-to-Charge (m/z)
[M+3H]3+
1.8
629.3407
423.2099
258.1558
100
y4
y3
[M+2H-2H3PO4]2+
a8
5
605.3256
b4
b2
R
R/P
b7
884.4540
y
y3
389.2520
0
136.0749
T a1
50
2
Fluoranthene Solid
y
1
0.5
276.1669
y1
1
0.6
1.5
[M+3H-H3PO4]3+
2.4
y2
1.2
0.8
3
2.6
476.2846
1.4
568.2544
3.5
2
y4
1.6
[M+3H]3+
6
5.5
[M+2H-3H3PO4]2+
983.5193
2
+ Product Ion (31.501-33.372 min, 15 scans) (568.2561[z=3] -> **) Phosphomix574CE3.9.d
7
4
QTOF CID MS/MS of VPIPGRFDRRVtVE
x10
A microfluidic-based nanoflow LC interfaced to an ion trap
mass spectrometer with ETD, or a QTOF mass
spectrometer were used to analyze the samples.
Fluoranthene was used as the chemical reagent for ETD.
All ETD and CID data were analyzed using Spectrum Mill
Protein Identification Workbench.
1366.1905
b8
2.4
4.5
+ Product Ion (16.653-19.204 min, 32 scans) (574.3000[z=3] -> **) Phosphomix574CE3.9.d
[M+2H-4H3PO4]2+
+ Product Ion (4.070-4.121 min, 2 scans) (1561.6532 -> **) tetra-msms-06.d
3
2.6
5
3
Phosphorylation site determination in ERK1
2.8
2.2
2.2
NCI
Source
Tetraphosphopeptide from β casein
QTOF CID MS/MS of ELEELNVPGEIVEsLsssEESITR
2.5
ETD instrumentation
Results and Discussion
Figure 3. QTOF CID, Ion trap CID and ETD spectra of
the phosphotyrosine peptide TRDIyETDYYRK. QTOF
CID produces low mass MS/MS fragments including
immonium ion for phosphotyrosine at 216Da. Ion
trap ETD provides the information of the exact
location of the phosphorylation.
Figure 4. QTOF CID, ion trap CID and ETD spectra of
ELEELNVPGEIVEsLsssEESITR, the beta-casein tetraphosphopeptide. QTOF CID produces four neutral
loss ions indicating there are four phosphorylation
sites. Ion trap ETD locates all four phosphorylation
sites on this peptide.
• Both QTOF and ion trap have demonstrated their
ability to characterize protein phosphorylation.
QTOF provides excellent mass accuracy and rich
low mass fragment ions including immonium ion
for phosphotyrosine.
• ETD preserves the PTMs on the amino acid during
the fragmentation, which allows the location of
the PTM to be determined.
• ETD and CID are complementary techniques. The
combination of both methods provides more
confident PTM characterization.