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OVERVIEW

An electron capture dissociation (ECD) cell that entraps low-

energy electrons from a heated filament using magnets and DC

electrostatic lens elements was installed within a modified

Synapt G2-Si .

The cell enables ECD before and after the travelling wave ion

mobility device of the Synapt G2-Si.

This research study will focus on results generated following

implementation of the ECD cell post ion mobility.

INTRODUCTION

Collision Induced Dissociation (CID) is the preferred dissociation

technique for tandem MS given its simplicity, ease of

implementation as well as reliable and robust performance for

many analytes. However, CID of large proteins, in particular non-

covalent complexes offers very little structurally informative

sequence ions. The product ions generated are typically formed

through sub-unit ejection of the intact complex. Electron Transfer

Dissociation (ETD) and Electron Capture Dissociation (ECD) are

known to provide sequence-specific information following

dissociation with little disruption of non-covalent interactions.

Unlike CID labile PTM’s are preserved during fragmentation and

sequence information is obtained for large proteins.

Structural Characterisation of Intact Proteins using Electron Capture Dissociation within an Ion Mobility enabled TOF

Jonathan P. Williams1, Lindsay Morrison2, Christopher J. Hughes1, Joseph Beckman3, Valery G. Voinov3, Frederik Lermyte4 and Jeffery M. Brown1 1Waters Corporation, Wilmslow, UK 2Waters Corporation, Beverly, USA 3e-MSion and Oregon University, Corvallis, USA 4Warwick University, Coventry, UK

Figure 2: Denatured Infusion ECD MS/MS of myoglobin (top) and ubiquitin (bottom). Following spectral deconvolution of the ECD data, a sequence coverage map of the product ions obtained from myoglobin 20

+ was provided from ProSight software. Inset tables represent sequence coverage and PC scores.

Figure 1: Schematic of the Synapt G2-Si and photograph following inclusion of the ECD cell post-ion mobility

Figure 4: Native hemoglobin tetramer (64kDa) (SEC-CIU IMS-ECD) with varying cone voltages. Spectra show lower m/z ECD product ions together with charge-reduced product ions at higher m/z. Note the quadrupole was operated in RF-only mode. Sequence coverage of both chains was determined using Masstodon soft-ware.

Figure 5: SEC-CIU IMS-ECD, Native NIST mAb (Fab 97kDa, SEC and IMS selected) following reaction with ideS. Low m/z ECD product ions together with high m/z charge-reduced product ions are observed in the spectrum. More low m/z ECD product ions are observed as unfolding occurs. Note the quadrupole was operated in RF-only mode.

CONCLUSIONS

• An electrostatic ECD was fitted to a modified Synapt for post ion mobility fragmentation.

• Intact proteins and non-covalent protein complexes were analyzed using size exclusion chromatography with ECD from eluting peaks of 10-20s (FWHM).

• Collision Induced Unfolding (CIU) combined with IMS and ECD allows surface mapping for location of protein unfolding.

• ECD of peptides is yet to be investigated.

• Improvements in overall protein sequence coverage is expected with the ECD cell posi-tioned pre-IMS since ion mobility will improve the peak capacity.

• The choice of the location of the ECD cell presents new experimental research possibili-ties.

METHODS

The e-MSion ECD cell is comprised of 7 electrostatic lenses, electrons are emitted from a hot loop rhenium fila-ment coated with ytrrium oxide. They are constrained by two samarium alloy magnets to the central axis which is aligned with the flight path of the ions exiting the ion mobili-ty cell. The standard transfer cell was shortened to accom-modate the ECD cell. For collision induced unfolding (CIU), ions are activated as

a function of either the source cone voltage or trap cell col-

lision energy within the Synapt G2-Si. CIU conformational

changes can be observed through ion mobility drift time

changes as unfolding occurs. Information relating to the

higher order structure of the protein may be obtained. In

addition, by recording the sequence ions generated by

ECD the location of unfolding can be mapped upon an X-

ray crystal structure (if available). Top-down and middle-

down ECD data is complex requiring accurate deconvolu-

tion. This is being investigated using software BayeSpray/

Unifi (Waters), BayeSpray/ProSight and IMTBX + Grppr/

Unifi/ProSight and MassTodon. The opportunity to perform

ECD pre-IMS will clearly improve product ion identification

for this analysis.

UPLC: H-Class Column: Acquity UPLC Protein BEH SEC Column (200Å, 1.7µM, 2.1mm x 150mm); Column Temperature: 30

oC; Sample Tempera-

ture: 8oC; Injection volume: 5µL

Mobile Phase: 100mM Ammonium Acetate with a flow rate of 100µL/min provided Isocratic Native Protein Elution MS: Synapt G2-Si with ECD modification; Ionisation: ESI (+VE) Capillary voltage: 2.1kV; Cone voltage: 75-150V; Source Tempera-ture: 50-110

oC; Desolvation Gas: 800L/hr

m/z200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400

%

0

100

m/z200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400

%

0

100942.7

942.6

893.0

893.0

892.9

892.9c''11 2+616.2c''5

447.2 618.2

942.8

942.9

942.9

998.1

998.3

1060.5

1062.5

1428.3

1427.6

1427.5c''6

764.5z'3

273.1 720.4

y''7 2+z'4

386.2c''10

1136.7

c''7865.5

1428.5 1713.9

c''45 3+1705.0

1704.8

2142.71718.3

2142.2 2143.0

2143.2

2143.4

2143.7

m/z1750 1800 1850 1900 1950 2000 2050

%

0

100

z'55 3+2073.4

1858.31747.5

c''47 3+1747.7

1825.7

1772.7

1951.3y''51 3+1926.1

1858.8

1872.8

1873.3

z'55 3+2072.9

1951.8

1952.1

1955.6

y''54 3+2044.6

2074.4

2074.7

2074.9

m/z840 845 850 855 860 865 870 875

%

0

100858.4

838.0 z'15 2+847.4

838.2

838.4

842.1

851.7858.0

866.1864.4

864.2

864.1

864.0

863.9

866.8c''24 3+870.1

874.4

Precursor

ion

Precursor ion

RESULTS

0

20

40

60

80

100

120

Bay

esSp

ray

IMTB

X

Bay

esSp

ray

IMTB

X

Bay

esSp

ray

IMTB

X

Bay

esSp

ray

IMTB

X

Myoglobin 20+

Myoglobin Pseudo MSE

Ubiquitin 12+ Ubiquitin Pseudo MSE

Seq

ue

nce

Co

vera

ge (

%)

TOF IM

0

200

400

600

800

1000

1200

1400

1600

1800

2000

Bay

esSp

ray

IMTB

X

Bay

esSp

ray

IMTB

X

Bay

esSp

ray

IMTB

X

Bay

esSp

ray

IMTB

X

Myoglobin 20+

Myoglobin Pseudo MSE

Ubiquitin 12+ Ubiquitin Pseudo MSE

PC

S

TOF IM

m/z20000 40000 60000 80000 100000

%

0

10025672.5

24819.0

23268.9

22560.6

21900.4

21270.5

20684.4

20124.3

19592.7

18618.3

14575.3

26588.3

27566.9

29771.7

31007.9

32361.9

33830.7 43776.4

46519.5

49603.6

57268.3

62019.4 68725.3 76363.685877.9

monomer

Intact14-mer

13-mer

12-mer

charge-reduced13-mer

charge-reduced12-mer

m/z5000 10000 15000 20000 25000 30000 35000 40000

%

0

1001917.1

1920.5

1983.7

2051.1

2054.4

m/z200 400 600 800 1000 1200 1400 1600 1800

%

0

1001054.4

924.3

834.5

502.3

403.2

288.2

824.8739.8

739.6

739.4630.4

824.5

920.5

920.1

1002.6

1236.7

1236.2

1135.6

1256.4

1336.8 1435.8 1581.1

1580.9

1581.9

1771.9

A) B)C)

m/z1000 2000 3000 4000 5000 6000 7000

%

0

100

m/z1000 2000 3000 4000 5000 6000 7000

%

0

100

m/z1000 2000 3000 4000 5000 6000 7000

%

0

100

m/z1000 2000 3000 4000 5000 6000 7000

%

0

100

m/z1000 2000 3000 4000 5000 6000 7000

%

0

1006509.36102.4

2464.12463.6

1968.61251.8

5743.7

5741.12464.9

6974.0

7510.6

7512.2

6509.26102.4

6101.52464.11251.8

727.52202.4

5742.2

6974.2

6974.9 7510.4

7512.1

6509.36102.6

6101.7

5741.51251.8

6974.0

7510.7

7511.9

6971.76509.0

6102.4

6099.0

5740.0

7510.6

7511.0

7512.3

6971.56509.46102.3

6096.8

7507.1

7511.4

7512.7

Time-0.00 2.50 5.00 7.50 10.00 12.50

%

0

100

-0.00 2.50 5.00 7.50 10.00 12.50

%

0

100

-0.00 2.50 5.00 7.50 10.00 12.50

%

0

100

-0.00 2.50 5.00 7.50 10.00 12.50

%

0

100

-0.00 2.50 5.00 7.50 10.00 12.50

%

0

1009.82

9.54

8.57

6.77

6.36

125V

100V

75V

50V

150V

Figure 3: A) MS spectrum of intact native GroEL (800kDa) with trap energy 100V, showing ion transmission with ECD cell in place (filament off). B) ECnoD (filament on) of quadrupole selected GroEL m/z 10,992 [M+73H]

73+ highlighting extreme charge-reduction and bimodal distribution C) ECD of quadrupole selected m/z 1909 [M+30H]

30+

from denatured GroEL monomer.

Figure 6: Sequence coverage for the ECD Fab LC portion was 27%, and the Fab Fd' portion was 16% with CV 125V. This does not include fragments that would include portions of both domains (which is the C-terminus of both because of a disulfide bond) For an analogous crystal structure, Green—LC sequence with increased fragmentation upon cone activa-tion and Blue HC sequence fragments at 50V and 125V cone activation.

ALPHA Chain 48% coverage (68/140 bonds)

31% coverage (45/145 bonds)

BETA Chain

m/z2000 4000 6000 8000 10000

%

0

100

m/z2000 4000 6000 8000 10000

%

0

100

m/z2000 4000 6000 8000 10000

%

0

100

m/z2000 4000 6000 8000 10000

%

0

1001225.7

829.51226.0

1505.8 6446.75860.7

1968.15111.64276.1 7163.0 8057.9

9207.9

656.9 5860.8829.5 5372.5

1235.24959.3

1310.8 4605.3

6446.8

7163.1 8057.89207.2

5861.15372.6

656.9829.5 4605.5

1042.64298.1

6446.9

7163.28057.5

9207.5

5372.34959.2

656.94605.1829.5

1309.7 4298.0

5861.0

6446.7

7163.18057.8

9208.2Time

-0.00 5.00 10.00 15.00

%

0

100

-0.00 5.00 10.00 15.00

%

0

100

-0.00 5.00 10.00 15.00

%

0

100

-0.00 5.00 10.00 15.00

%

0

10011.12

9.84

7.84

7.84

100V

75V

50V

25VDrift time ms

filament