Ion Mobility Mass Spectrometry of Native Protein Complex...
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Research Royalty Fund The Richard A. Schaeffer Memorial Fund Key Outcomes • Ion mobility (IM) mass spectrometry used to characterize pro- tein complex anions. • Anions adopt smaller average charge states than the corre- sponding cations, which may be consistent with lower field- emission energies for the loss of charge carriers from anions. • Triethylamine addition results in the appearance of additional, charge-reduced cations. • Cations, anions, and charge-reduced cations all have similar collision cross section (Ω) values, suggesting that they all have similar structures that depend weakly on charge state. • These anion Ω values can be used to measure accurate cali- brated Ω values with traveling-wave IM. Drift times for anions of native-like proteins were measured at 10 drift voltages ranging from 100 - 350 V in 2 Torr of helium gas. An RF-confining drift tube has been implemented in place of the traveling-wave ion mobility cell on a Waters Synapt G2 HDMS, which controls all potentials and regulates gas flow. Traveling-wave IM was used to determine Ω values (Ruotolo et al. Nat. Prot. 2008, 3, 1139) of selected protein complexes. For ex- ample, pyruvate kinase anion drift times (black) were calibrated using literature cation Ω values (red). Calibration errors are evi- dent from the observed deviations from the calibration lines. Anion drift times calibrated using anion Ω values ( ) are more ac- curate than those calibrated using cation Ω values ( ). Similarly, cation drift times calibrated using cation Ω values ( ) are more accurate than those calibrated with anion Ω values ( ). Charge Reduction Measuring Collision Cross Sections (Ω) Ion Mobility Mass Spectrometry of Native Protein Complex Anions Samuel J. Allen, Alicia M. Schwartz, Matthew F. Bush | University of Washington, Seattle, WA Charge States Field-Emission Model Thanks Cation vs. Anion Ω Values (Top) Ω values of charge-reduced cations (blue) are compared to those of the orignal cations (red) and anions (black). Cation Ω values measured with and without triethylamine are indistin- guishable. (Bottom) The differences between the Ω values of charge- reduce cations and anions of the same |z| are small. Here, we observed that anions of protein complexes formed by nanoelectrospray ionization have significantly smaller average charge states than the corresponding cations. This result is con- sistent with anions having lower field-emission energies for the loss of charge carriers. Interestingly, cations, anions, and charge- reduced cations all have similar Ω values, suggesting that they all have similar structures that depend weakly on charge state. We also demonstrated that these Ω values can be used to measure accurate calibrated Ω values with traveling-wave IM. Conclusions Anion Traveling-Wave Calibration Dual Stage Reflectron High-Field Pusher Detector Transfer Cell 10 –2 mBar See Inset Trap Cell 10 –2 mBar Ion Guide 10 –3 mBar Quad 10 –5 mBar Time-of-Flight Mass Analyzer Time-of-Flight Mass Analyzer 10 –7 mBar Nano ESI 1 atm. RF(–) RF(+) RF-Confining Drift Tube 25.2 cm | 84 Plate Pairs | 2-5 mBar He or N 2 | 5 mm ID 330 pF capacitor 10 MΩ resistor 5 MΩ resistor V in V out Distance V in V out DC Potential entrance exit The addition of 10 mM triethlyamine to the protein solution re- sults in the appearance of a much wider charge-state distribu- tion. Charge-reduced avidin and β-galactosidase (blue) are shown below. The original positive (red) and negative (black) ion mode spectra are shown as a guide to the eye. 8k 14k 0 300 100 200 400 Mass / kDa 40 30 20 10 Average Charge State | 〈 z 〉 | 500 50 Cations Anions Avidin β-galactosidase 3k 6k 4k 5k m/z Avidin Cations Anions +17 +16 –14 –13 10k 12k m/z β-galactosidase Cations Anions +48 +49 –39 –38 〈 z 〉 = +16.4 〈 z 〉 = +48.8 〈 z 〉 = –13.4 〈 z 〉 = –38.4 Hogan et al. Anal. Chem. 2009, 81, 369-377. z ∝ E* (Diameter) 2 X + X + Y + Y + X + Z + Z + X + Y + X + Z + Y + Y + X + X + Y + X + X + X + X + + + + + Z + Y + Z + Y + Y + X + X + X + X + E* Z + E* Y + E* X + 1 / Drift Voltage 0.01 0.002 0.004 0.006 0.008 2 4 6 8 10 t 0 1.98 9.92 4.96 3.31 2.48 Drift Field / V cm -1 Drift Time / ms K 0 ∝ z(1/μ) 1/2 (1/ Ω) 12 14 β-galactosidase Pyruvate Kinase Concanavalin A Avidin Ubiquitin Initial ESI Droplets nanoDroplets formed via Coulomb fission CRM-based evap. ze= 8π(ε 0 γ(D/2) 3 ) 1/2 Wave Height = 24 V Wave Height = 32 V Wave Height = 28 V 140 110 80 50 Ω / nm 2 20 25 30 35 Corrected Drift Time / t D ” 40 45 50 55 glut. dehyd. cations alcohol dehyd. cations pyruvate kinase anions Voltage Axial Radial +15 –12 +18 +47 +50 –37 –40 –36 4 2 0 -2 -4 -6 -8 2000 6000 8000 14000 m/z Ω % Difference avidin bov. serum albumin alcohol dehyd. pyruvate kinase glut. dehyd. β-galactosidase 160 120 80 40 Ω / nm 2 Cations Anions Charge-Reduced Cations ubiquitin β-galactosidase avidin cytochrome c pyruvate kinase bov. serum albumin glut. dehyd. alcohol dehyd. β-lacto. monomer β-lacto. dimer Ω Anion > Ω Cation Ω Anion < Ω Cation 24 26 28 Charge State |z| 30 32 34 36 10 6 2 -2 % Error fo pyruvate kinase 8 4 0 Calibrant Ions Cations Anions [PK] + [PK] – Ω T-wave > Ω Literature Ω T-wave < Ω Literature E* = 2.40 V nm -1 E* = 3.53 V nm -1 E* = 2.95 V nm -1 Rayleigh Limit never approached –10 –3 Average Charge State 〈 z 〉 Diameter / nm –50 3 10 10 2 5 This work This work Hogan et al. E* = 1.86 V nm -1 E* = 2.75 V nm -1 E* = 2.22 V nm -1 50 Cations Anions 3k 6k 4k 5k m/z 8k 14k 10k 12k m/z +14 +13 +17 +16 +39 +48 +49 Avidin β-galactosidase +15 +11 +12 +50 +51 +47 +37 +36 +46 +40 +38 Field Emission F electric F drag Anions adopt smaller average charge states than cations Rayleigh Limit Rayleigh Limit Rayleigh Limit approached at high mass Extremely Linear Response Identity critical, z subtle Trends subtle, under investigation
Transcript of Ion Mobility Mass Spectrometry of Native Protein Complex...
Research Royalty Fund
The Richard A. Schaeer Memorial Fund
Key
Out
com
es
• Ion mobility (IM) mass spectrometry used to characterize pro-tein complex anions.
• Anions adopt smaller average charge states than the corre-sponding cations, which may be consistent with lower field-emission energies for the loss of charge carriers from anions.
• Triethylamine addition results in the appearance of additional, charge-reduced cations.
• Cations, anions, and charge-reduced cations all have similar collision cross section (Ω) values, suggesting that they all have similar structures that depend weakly on charge state. • These anion Ω values can be used to measure accurate cali-brated Ω values with traveling-wave IM.
Drift times for anions of native-like proteins were measured at 10 drift voltages ranging from 100 - 350 V in 2 Torr of helium gas.
An RF-conning drift tube has been implemented in place of the traveling-wave ion mobility cell on a Waters Synapt G2 HDMS, which controls all potentials and regulates gas ow.
Traveling-wave IM was used to determine Ω values (Ruotolo et al. Nat. Prot. 2008, 3, 1139) of selected protein complexes. For ex-ample, pyruvate kinase anion drift times (black) were calibrated using literature cation Ω values (red). Calibration errors are evi-dent from the observed deviations from the calibration lines.
Anion drift times calibrated using anion Ω values ( ) are more ac-curate than those calibrated using cation Ω values ( ). Similarly, cation drift times calibrated using cation Ω values ( ) are more accurate than those calibrated with anion Ω values ( ).
Char
ge R
educ
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Mea
suri
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ollis
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Cros
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ns (Ω
)Ion Mobility Mass Spectrometry of Native Protein Complex Anions
Samuel J. Allen, Alicia M. Schwartz, Matthew F. Bush | University of Washington, Seattle, WA
Char
ge S
tate
sFi
eld-
Emis
sion
Mod
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Than
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Cati
on v
s. A
nion
Ω V
alue
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(Top) Ω values of charge-reduced cations (blue) are compared to those of the orignal cations (red) and anions (black). Cation Ω values measured with and without triethylamine are indistin-guishable.(Bottom) The dierences between the Ω values of charge-reduce cations and anions of the same |z| are small.
Here, we observed that anions of protein complexes formed by nanoelectrospray ionization have signicantly smaller average charge states than the corresponding cations. This result is con-sistent with anions having lower eld-emission energies for the loss of charge carriers. Interestingly, cations, anions, and charge-reduced cations all have similar Ω values, suggesting that they all have similar structures that depend weakly on charge state. We also demonstrated that these Ω values can be used to measure accurate calibrated Ω values with traveling-wave IM.
Conc
lusi
ons
Ani
on T
rave
ling-
Wav
e Ca
libra
tion
Dual Stage Reflectron
High-FieldPusher
DetectorTransfer
Cell10–2 mBar
See InsetTrapCell
10–2 mBar
IonGuide
10–3 mBar
Quad10–5 mBar
Time-of-FlightMass AnalyzerTime-of-FlightMass Analyzer
10–7 mBar
NanoESI
1 atm.
RF(–) RF(+)
RF-Confining Drift Tube25.2 cm | 84 Plate Pairs | 2-5 mBar He or N2 | 5 mm ID
330 pF capacitor
10 MΩ resistor
5 MΩ resistor
Vin Vout
Distance
Vin
Vout
DC
Pote
ntia
l
entrance
exit
The addition of 10 mM triethlyamine to the protein solution re-sults in the appearance of a much wider charge-state distribu-tion. Charge-reduced avidin and β-galactosidase (blue) are shown below. The original positive (red) and negative (black) ion mode spectra are shown as a guide to the eye.
8k 14k
0 300100 200 400Mass / kDa
40
30
20
10
Ave
rage
Cha
rge
Stat
e | 〈
z 〉|
500
50
Cations
AnionsAvidin
β-galactosidase
3k 6k4k 5km/z
Avidin
Cations
Anions
+17 +16
–14 –13
10k 12km/z
β-galactosidase
Cations
Anions
+48+49
–39 –38
〈z〉 = +16.4 〈z〉 = +48.8
〈z〉 = –13.4 〈z〉 = –38.4
Hogan et al. Anal. Chem. 2009, 81, 369-377.
z ∝ E* (Diameter)2
X+
X+
Y+
Y+
X+Z+
Z+
X+
Y+
X+
Z+
Y+
Y+
X+ X+Y+X+
X+X+
X+
+
+
++
Z+Y+ Z+
Y+Y+X+
X+
X+ X+
E*Z+
E*Y+E*X+
1 / Drift Voltage0.010.002 0.004 0.006 0.008
2
4
6
8
10
t0
1.989.92 4.96 3.31 2.48Drift Field / V cm-1
Dri
ft T
ime
/ ms
K0 ∝ z(1/μ)1/2(1/ Ω)
12
14β-galactosidasePyruvate Kinase
Concanavalin AAvidinUbiquitin
Initial ESIDroplets
nanoDroplets formed via Coulomb fission
CRM-based evap.ze= 8π(ε0γ(D/2)3)1/2
Wave Height = 24 V
Wave Height = 32 VWave Height = 28 V
140
110
80
50
Ω /
nm2
20 25 30 35Corrected Drift Time / tD”
40 45 50 55
glut. dehyd. cations
alcohol dehyd. cations
pyruvate kinase anions Voltage
Axial Radial
+15
–12
+18+47+50
–37–40
–36
4
2
0
-2
-4
-6
-8
2000 6000 8000 14000m/z
Ω %
Di
eren
ceavidinbov. serum albuminalcohol dehyd.pyruvate kinaseglut. dehyd.β-galactosidase
160
120
80
40
Ω /
nm2
CationsAnionsCharge-Reduced Cations
ubiquitin
β-galactosidase
avidin
cytochrome c
pyruvate kinase
bov. serum albumin
glut. dehyd.
alcohol dehyd.
β-lacto. monomer
β-lacto. dimer
ΩAnion > ΩCation
ΩAnion < ΩCation
24 26 28Charge State |z|
30 32 34 36
10
6
2
-2% E
rror
fo p
yruv
ate
kina
se
8
4
0
Calibrant IonsCations Anions
[PK]+[PK]–
ΩT-wave > ΩLiterature
ΩT-wave < ΩLiterature
E* = 2.40 V nm-1
E* = 3.53 V nm-1
E* = 2.95 V nm-1
Rayleigh Limit never approached
–10
–3
Ave
rage
Cha
rge
Stat
e 〈z 〉
Diameter / nm
–503
10
102 5
This work
This workHogan et al.
E* = 1.86 V nm-1
E* = 2.75 V nm-1
E* = 2.22 V nm-1
50Cations
Anions
3k 6k4k 5km/z
8k 14k10k 12km/z
+14
+13+17
+16
+39
+48+49
Avidin β-galactosidase
+15
+11
+12
+50
+51
+47
+37+36
+46+40
+38
Field Emission
FelectricFdrag
Anions adopt smaller average charge states than cations
Rayleigh Limit
Rayleigh Limit
Rayleigh Limit approached at high mass
Extremely Linear Response
Identity critical, z subtle
Trends subtle, under investigation
