Supporting Information:
Examining Factors that Influence Erroneous Phosphorylation Site Localization via
Competing Fragmentation and Rearrangement Reactions during Ion Trap CIDMS/MS and -MS3
Li Cui1 and Gavin E. Reid1,2*
1
2
Department of Chemistry, Michigan State University, East Lansing, MI 48824
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI
48824
*Corresponding Author
Michigan State University
578 S. Shaw Lane. Chemistry Building, Room 229
East Lansing, Michigan
USA, 48824
Phone: (517)-355-9715 x198; Fax: (517)-353-1793.
Email: reid@chemistry.msu.edu
1
Supplemental Materials and Methods
Materials
All amino acid derivatives and preloaded Wang-resins were purchased from EMD bioscience
(Novabiochem)
(Hohenbrunn,
Germany).
O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TBTU) and N-Hydroxybenzotriazole (HOBt) were from Peptides International
(Louisville, KY). 2,5-DHB (HPLC grade), piperidine (biotech grade), N,N-diisopropylethylamine
(DIPEA, biotech grade) and triisopropylsilane (99%) were purchased from Sigma Aldrich (St. Louis,
MO). Dichloromethane (DCM, 99.5%), acetic acid (99.7%) and acetic anhydride (97%) were from
Mallinckrodt Chemicals (Phillipsburg, NJ). DMF (99.8%) was purchased from Jade Scientific (Canton,
MI) and stored in 4Å molecular sieves. TFA (LC grade) was from Thermo Scientific (San Jose, CA).
Methanol (99.8%) was from VWR BDH chemicals (West Chester, PA). ACN (HPLC grade) was
purchased from EMD Chemicals (Gibbstown, NJ). All peptide solutions were prepared using deionized
water purified by a Barnstead nanopure diamond purification system (Dubuque, IA).
Mass Spectrometry
Crude peptides were individually dissolved in 50% methanol, 1% acetic acid (v/v) at a
concentration of 20 μM. For MALDI analysis, 0.25 μL of above peptide solution was directly mixed on
the plate with 0.25 μL of a 2,5-DHB matrix solution (50 mg/mL in 1:1 acetonitrile:H2O), then allowed
to dry prior to introduction to the mass spectrometer. For ESI analysis, samples were loaded into
Whatman multichem 96-well plates (Sigma Aldrich, St. Louis, MO) and sealed with Teflon Ultra Thin
2
Sealing Tape (Analytical Sales and Services, Prompton Plains, NJ). Then, 13 μL of the peptide solutions
were introduced to the mass spectrometer by direct infusion using an Advion BioSciences Triversa
Nanomate nESI source (Ithaca, NY). Both MALDI and nESI conditions were optimized to maximize
the sensitivity and stability of the precursor ions of interest while minimizing ‘in-source’ fragmentation.
The Advion spray voltage was maintained at 1.4 kV and the gas pressure was set at 0.3 psi. The ion
transfer tube of the mass spectrometer was set at 180°C for the LTQ and at 250°C for the LTQ Orbitrap
Velos and the applied ESI voltage was 2 kV. The S-Lens was set to 47% for LTQ Orbitrap Velos.
Isolation widths for the singly, doubly and triply protonated precursor ions were 3.0, 2.5 and 2.0
respectively. Ion activation conditions were activation q = 0.25 and normalized collision energy (NCE)
35%, using a default activation time of 30 ms for the LTQ XL and LTQ, and a default activation time of
10 ms for the LTQ Orbitrap Velos. The LTQ Orbitrap Velos MS/MS spectra were acquired using a
mass analyzer resolution of 60,000. Depending on precursor ion abundances, spectra were acquired for
an average of 350-1000 scans.
3
Supplemental Schemes
A
B
H3PO4 loss
HPO3 and/or H2O loss
C
HPO3 transfer
amide bond cleavage
Scheme 1.
Competing pathways for the CID-MS/MS gas-phase fragmentation reactions of
protonated phosphopeptide ions. (A) Competition between H3PO4 loss side chain fragmentation and
amide bond cleavage pathways, (B) competition within side chain fragmentation pathways involving the
loss of H3PO4 or the combined losses of HPO3 and H2O, and (C) intramolecular phosphate group
(HPO3) transfer (i.e., rearrangement or ‘scrambling’).
4
Supplemental Figures
A
Relative Abundance
100
Average HPO3
rearrangement: 11.0%
-□2+
b8
o
b8
b6
4.5%
10.9%
17.5%
b4o
a6
b8■
■
■
y
b6 a
b4
8
8
y b4 y4
y
b6o
b3
3
6
600
B
G R A S P V P A P pY G G L H A A V R
y12
y14 y12 y10
23.3%
60.7%
y14□ y12□ y10□
□
y
10.2%
12
y14□
-oo2+
□o
o
y
y
□
□
12
y14
y10
y14o 14
y15
y12o b
y10□o y10o b11
y16 b17
b14 b15
13
1000
1400
1200
-□o2+
[M+2H]2+
LTQ-CID
b4■ b6■ b8■
b4 b6 b8
Average HPO3 loss /
rearrangement: 31.4%
y10
800
x5
100
x5
- □oo2+
[M+2H]2+
LTQ-CID
400
Relative Abundance
-□o2+
x5
1600
1800
2000
x5
-□2+
b4
Average HPO3 loss: 4.7%
b6 b8
G R A A P V P A P pY G G L H A A V R
-□oo2+
y12
a6
b4
b3 a4
y4 b
400
b8
b6
y102+
y122+
a8
y8
y6
5
600
800
y14 y12 y10
4.4%
y14□ y12□ y10□
6.6%
3.2% y10 y12□
□
y
y10□
14
y12□o
o
b10
y12o y14□o y14
b
o
□
y10
y14 y15 16
y10o b11
y16 b17
b14 b15
1000
1200
1400
1600
1800
2000
m/z
Figure S1. ESI-LTQ ion trap CID-MS/MS product ion spectra from the doubly protonated precursor
ions
of
the
phosphopeptides
(A)
GRASPVPAPpYGGLHAAVR
and
(B)
GRAAPVPAPpYGGLHAAVR. ■ = +80 Da (+HPO3), ∆ = –98 Da (–H3PO4 or –(H2O+HPO3)); □ = –
80 Da (–HPO3); ° = –18 Da (–H2O).
5
A
Relative Abundance
100
b17●2+
-282+
-422+
[M+2H]2+
b16●2+
LTQ-CID
[M+2H-98]2+
b152+ b162+
y122+ b6
b4*
y3o
y5o
-302+
a6 b6*
b8
b4
600
b17
y12
b13●
y10
800
B
Relative Abundance
-o2+
b15●2+
400
100
G R A pY□ P V P A P So G G L H A A V R
b172+
b13
1000
1200
b14
y14
b14● b
●
15
b15
b16
b17●
b16●
1400
1600
b17●♦
1800
2000
b17●2+
-282+
-422+
[M+2H]2+
G R A So P V P A P pY□ G G L H A A V R
b16●2+
LTQ-CID
[M+2H-98]2+
b172+
-*2+
b15●2+
b152+
b6
b13
b162+
-302+
y10o
b6
●
y12o
y10
y3o
400
600
800
1000
b17
b14● y
14
b
y12b13● y o b15 16
14
b17●
●
b
15
b14
b16●
1200
1400
1600
b17●♦
1800
2000
m/z
ESI-LTQ ion trap CID-MS3 product ion spectra for the doubly protonated [M+2H-98]2+
Figure S2.
neutral
loss
product
ions
from
the
doubly
protonated
phosphopeptides
(A)
GRApYPVPAPSGGLHAAVR from Figure 1A and (B) GRASPVPAPpYGGLHAAVR from
Supplemental Figure 1A. ● = +18 Da (–H2O); ♦ = +17 Da (+NH3); □ = –80 Da (–HPO3); ° = –18 Da (–
H2O); ); * = –17 Da (–NH3).
6
A
Relative Abundance
100
-∆2+
x5
Average HPO3
rearrangement: 4.8%
Average HPO3 loss /
rearrangement: 21.6%
16.4%
b4∆ b4□
y4 b4
G R A pS P V P A P S G G L H A A V R
y12
17.1% 31.3% ∆o2+ -□2+
y14
□
□
b
b
o
8
6
o
y
5.0%
8.0%
∆
b
10
y
1.5%
8
b6
14
b6
y10 y10■
y5
y12■
y14■
b8∆
600
800
B
Relative Abundance
b4□ b6□ b8□
b4 b6 b8
LTQ-CID
400
100
x5
[M+2H]2+
1000
1200
1400
y14 y12 y10
y14■ y12■ y10■
b17∆
b17
1600
1800
2000
b17●2+
[M+2H]2+
b172+
LTQ-CID
[M+2H-98]2+
-o2+
-282+
b16●2+
-302+
b162+
b152+
b4
G R A pSΔ P V P A P S G G L H A A V R
-422+
y12
b15●2+
b8
b8
b6
●
b4●
b6●
400
600
800
y10o
y10
y12o
1000
y14o
b
b y16 17
y14 b15● 16
b17●
b16●
b17●♦
b
b14●
b14
1200
15
1400
1600
-MS3
product
1800
2000
m/z
Figure S3.
ESI-LTQ
ion
trap
CID-MS/MS
and
ion
spectra
of
GRApSPVPAPSGGLHAAVR. (A) CID-MS/MS of the doubly protonated precursor ion and (B) CIDMS3 of the [M+2H-98]2+ neutral loss product ion from panel A. ■ = +80 Da (+HPO3); ● = +18 Da (–
H2O); ♦ = +17 Da (+NH3); ∆ = –98 Da (–H3PO4 or –(H2O+HPO3)); □ = –80 Da (–HPO3); ° = –18 Da (–
H2O).
7
GRApYPVPAPSGGLHAAVR
GRApYPVPAPTGGLHAAVR
GRApYPVPAPYGGLHAAVR
GRApTPVPAPSGGLHAAVR
GRApTPVPAPTGGLHAAVR
GRApTPVPAPYGGLHAAVR
GRApSPVPAPSGGLHAAVR
GRApSPVPAPTGGLHAAVR
GRApSPVPAPYGGLHAAVR
GRApYPVPAPSGGLHAAVK
GRApYPVPAPSGGLHAAVG
GKApYPVPAPSGGLHAAVR
GGApYPVPAPSGGLHAAVR
0
5
10
15
20
Average Rearrangement Ratio (%)
Figure S4.
Average % phosphate group rearrangement for the doubly protonated ([M+2H] 2+)
precursor ions of the synthetic phosphopeptide library GX1AX2PVPAPX3GGLHAAVX4, where X1 =
R, K or G; X2 = pY, pT or pS; X3 = S, T or Y and X4 = R, K or G, acquired by ion trap CID-MS/MS
using ESI-LTQ (dark blue) or ESI-LTQ Velos (light blue) mass spectrometers. Average rearrangement
ratios were calculated only from the observed yn■, yn■■, yn product ions. Error bars show the standard
deviation of the individual % phosphate group rearrangements determined from each product ion.
8
A
B
LFpSGHPESLER
LFpSGHPETLER
LFpSGHPEYLER
LFpTGHPESLER
LFpTGHPETLER
LFpTGHPEYLER
LFpYGHPESLER
LFpYGHPETLER
LFpYGHPEYLER
0
1
2
3
4
5
6
GpSpSQELDVKPSASPQER
GpSpSQELDVKPTATPQER
GpSpSQELDVKPYAYPQER
GpTpTQELDVKPSASPQER
GpTpTQELDVKPTATPQER
GpTpTQELDVKPYAYPQER
GpYpYQELDVKPSASPQER
GpYpYQELDVKPTATPQER
GpYpYQELDVKPYAYPQER
C
pSPLPAPPRPFLSR
pSPLPAPPRPFLTR
pSPLPAPPRPFLYR
pTPLPAPPRPFLSR
pTPLPAPPRPFLTR
pTPLPAPPRPFLYR
pYPLPAPPRPFLSR
pYPLPAPPRPFLTR
pYPLPAPPRPFLYR
0
0
10
20
30
40
10
20
30
40
Average Rearrangement Ratio (%)
Figure S5.
Average % phosphate group rearrangement for the doubly protonated ([M+2H]2+)
precursor ions of several synthetic phosphopeptide libraries analyzed by ESI-LTQ CID-MS/MS.
Average ratios were calculated from all y■, y■■ and b□, b□□ product ions. (A) y4■, y6■, y7■, y8■, b4□, b3□,
b4□, b5□ and b7□ ions were observed for the LFX1GHPEX2LER peptides (where X1 = pS, pT or pY, and
9
X2 = S, T or Y), (B) y8■/y8■■,y9■/y9■■, y10■/y10■■, y11■/y11■■, y12■/y12■■, y13■/y13■■, b5□/ b5□□, b6□/ b6□□,
b7□/ b7□□, b8□/ b8□□ and b9□/ b9□□ ions were observed for the GX1QELDVKPX2AX2PQER peptides
(where X1 = pSpS, pTpT or pYpY, and X2 = S, T or Y) and (C) y8■, y10■, b3□, b5□and b8□ ions were
observed for the X1PLPAPPRPFLX2R peptides (where X1 = pS, pT or pY, and X2 = S, T or Y). Error
bars show the standard deviation of the individual % phosphate group rearrangements determined from
each of product ion.
10
GRApYPVPAPSGGLHAAVR
GRApYPVPAPTGGLHAAVR
GRApYPVPAPYGGLHAAVR
GRApTPVPAPSGGLHAAVR
GRApTPVPAPTGGLHAAVR
GRApTPVPAPYGGLHAAVR
GRApSPVPAPSGGLHAAVR
GRApSPVPAPTGGLHAAVR
GRApSPVPAPYGGLHAAVR
GRApYPVPAPSGGLHAAVK
GRApYPVPAPSGGLHAAVG
GKApYPVPAPSGGLHAAVR
GGApYPVPAPSGGLHAAVR
0
20
40
60
80
100
Average Rearrangement Ratio (%)
Figure S6.
Average % phosphate group rearrangement for the singly protonated ([M+H] +) precursor
ions of the synthetic phosphopeptide library GX1AX2PVPAPX3GGLHAAVX4, where X1 = R, K or
G; X2 = pY, pT or pS; X3 = S, T or Y and X4 = R, K or G, acquired by MALDI CID-MS/MS using
ESI-LTQ (dark blue) or ESI-LTQ Velos (light blue) mass spectrometers. Average rearrangement ratios
were calculated from the observed yn■, yn■■, yn, and bn□, bn□□, bn product ions. Error bars show the
standard deviation of the individual % phosphate group rearrangements determined from each product
ion.
11
x10
A
x10
100
Relative Abundance
-∆2+
-∆∆2+
LTQ-CID
-∆∆o2+
600
y12
y14
27.3%
13.2%
■
y12 y o
14
48.0%
16.4% y15 y15■
y10■ y12■o
y14■
b15 b16
y10■o
y10
800
pS P V P A P S S S L H A A V R
y15 y14 y12 y10
y15■ y14■ y12■ y10■
-oo2+
b7□□ b9∆□ b □
9
b5∆□ b □□
∆
□
5
b7□
b7
b
b4∆ b5∆∆
7
∆
b4 b5□ b7∆∆ b7
b9∆∆ b9□□
b4□
y6 y
y8
y4
b5∆ y5
y3
7
b4□
b4
G R A pS
b9
400
y12o
1000
1200
1400
1600
b18
y17
1800
2000
x10
B
-∆2+
x10
100
Relative Abundance
Average rearrangement:
32.3%
[M+2H]2+
b5∆□ b7∆□ b9∆□
b5□□ b7□□ b9□□
b5□ b7□ b9□
b5 b7 b9
Average rearrangement:
40.3%
[M+2H]2+
-oo2+
LTQ-CID
b7∆ b7
b5∆□
b4∆
b5∆∆
□
y3 b4
y4
400
Figure S7.
b9∆□ -∆∆2+
b7∆□
y5
b9□
b7□□ □
b7
b4
b7∆∆
∆∆
y6
b9
b5∆ b □
y7
5
600
y12
-∆□2+
800
b5□□b7□□ b9□□
b4□ b5□ b7□ b9□
b4 b5 b7 b9
G R A pS
pS P V P A P S S S L H A A V K
y15 y14 y12 y10
y15■ y14■ y12■ y10■
y12o
24.1% y14
y12■ y o
2.9%
14
93.5%
14.7%
y15■
y10 y10■ y12■o
y14■ y15 b15 b
16
y10■o
1000
m/z
1200
1400
1600
y17
1800
2000
ESI-LTQ ion trap CID-MS/MS of the doubly protonated precursor ion from the doubly
phosphorylated
peptides
(A)
GRApSpSPVPAPSSSLHAAVR
and
(B)
GRApSpSPVPAPSSSLHAAVK. Average rearrangement ratios were calculated from the observed
yn■, yn■■, yn, and bn□, bn□□, bn product ions. ■ = +80Da (+HPO3); ∆ = –98Da (–H3PO4 or –(H2O+HPO3));
□ = –80Da (–HPO3); ° = –18Da (–H2O).
12