ion mobility mass spectrometry of native protein complex...
TRANSCRIPT
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
tion
Mea
suri
ng C
ollis
ion
Cros
s Se
ctio
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
el
Than
ks
Cati
on v
s. A
nion
Ω V
alue
s
(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