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