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INTRODUCTION

The use of mass spectrometry in the characterisation of high

molecular weight species has been an area of considerable

growth over the past ten to fifteen years. Key areas of

investigation have been on non-covalently bound protein

complexes of many hundreds of kilodaltons to virus capsids of

many megadaltons. An area of more recent and significant

growth has been in biopharmaceutical characterisation. The

high mass capability of time-of flight analysers has made them

the instrument of choice for the study of such systems. Here

we investigate the high mass performance characteristics of

quadrupole/ion mobility/time-of-flight instrument (Q-IM-TOF)

incorporating a novel conjoined source ion guide.

PROBING THE HIGH MASS TRANSMISSION CHARACTERISTICS OF A CONJOINED ION GUIDE

ENABLED QUADRUPOLE / ION MOBILITY / TIME-OF-FLIGHT INSTRUMENT

Kevin Giles, Jason Wildgoose, Jonathan Williams and Martin De Cecco

Waters Corporation, Manchester, UK

The larger diameter (15 mm) ion guide is aligned with the ion/

gas flow exiting the source region, see Figure 3. A DC

potential difference between this guide and the second,

smaller diameter (5 mm), ion guide extracts and focuses the

ions from the main gas stream into the second guide. The ion

optical axis of the second ion guide is aligned with the ion

optical axes of the subsequent ion guide and the mass

analysers. This design simultaneously concentrates the ion

beam from the diffuse source and reduces the degree of

contamination from neutral material entrained in the gas.

The conjoined ion guide operates at a pressure of about 3-4 mb

with variable RF amplitudes up to 320 V peak-to-peak at 1 MHz.

The DC offset between the conjoined ion guides is typically set at 25

V. In these experiments, the effect of pressure, RF and DC offset

on the transmission of three high mass species is investigated. All

samples were infused using borosilicate glass nano-electrospray tips

and analysed in positive ion mode. Data were acquired in TOF MS

or TOF MS/MS sensitivity mode (resolution ~15,000 FWHM) with

electrospray voltages of around +1.5 kV used throughout and

sample cone voltages of up to 200 V employed for de-clustering.

Trap and transfer collision cell pressures of 2-4x10-2 mb argon were

used with trap collision voltages in the 20-40 V range (except as

noted for MS/MS). The detector voltage was also increased for best

performance with these high mass species. The source backing

pressure on the Synapt G2 was optimised at ~7 mb.

Samples

The samples used in these studies were a mouse monoclonal

antibody (MAB) (mw ~150 kDa (Waters, 186006552)), Glutamate

Dehydrogenase Hexamer (GDH) (mw 336 kDa (Sigma, G7882)) and

GroEL (mw 802 kDa (Sigma, C7688)). All samples were buffer

exchanged into 200 mM/100 mM aqueous ammonium acetate (MAB

and GDH/GroEL) to a final concentration of 1 mg/mL.

RESULTS AND DISCUSSION

The mass spectra obtained for the three species using the Synapt

G2-S instrument are shown below in Figs 4, 5 and 6.

OVERVIEW

Investigation of the high mass transmis-

sion properties of a conjoined ion guide

system

Studies undertaken on Synapt G2 and

Synapt G2-S quadrupole/ion mobility/

time-of-flight instruments

Excellent transmission observed with

consistent increases in signal compared

with standard ion guide arrangement

METHODS

Instrumentation

The primary instrument used for this work was a Synapt G2-S

(Waters Corp.) which is shown schematically in Figure 1,

where the dual ion guide arrangement in the source transfer

region can be seen. Also shown in Figure 1 is the source

region of a Synapt G2, highlighting the presence of a single ion

guide between the source and analyser. The conjoined ion

guide in the Synapt G2-S consists of two different diameter

stacked ring electrode devices which are radially offset, as

shown in Figure 2.

Figure 1 A schematic diagram of the Synapt G2-S mass spectrometer

and the Synapt G2 source region.

As can be seen, intense mass spectral peaks are obtained from

these samples, even at 1 second acquisition times with essentially

equivalent data obtained using analogue- or time-to-digital (ADC

or TDC) acquisition modes.

Conjoined Guide Transmission Characteristics

The transmission characteristics of the conjoined ion guide as a

function of applied RF voltage, DC offset between the conjoined

ion guides and pressure in the conjoined ion guide are shown in

Figures 7, 8 and 9 respectively. The three high mass species

show broadly similar transmission characteristics as a function of

applied RF and DC. The transmission characteristic of singly

charged verapamil (m/z 455) is also plotted for comparison. The

RF characteristics of the low and high mass species are as

expected but the similarity of response to DC offset over the wide

m/z range is perhaps surprising. The pressure in the conjoined

ion guide region was increased from the ~4 mb base pressure by

throttling the backing pump line using a Speedivalve. It can be

seen that there is little or no effect of increased pressure on GDH

transmission, unlike that of other systems where some degree of

source pressure optimisation is necessary.

Synapt G2-S System Performance

To compare the relative high mass transmission efficiency of the

conjoined ion guide with that of the single ion guide, a series of

back-to-back experiments were performed on adjacent Synapt

G2 and G2-S instruments using the same sample. An initial

comparison was undertaken using the fragments of the doubly

charged Glu-fibrinopeptide B (GFP) ion, as shown in Figure 10.

The observed factor of ~25 increase in transmission using the

conjoined ion guide is as expected from previous studies. The

results for the high mass species are shown in Figure 11. The

signals on the conjoined ion guide system (G2-S) are

consistently higher than those on the single ion guide system

(G2). Absolute comparisons between instruments was

challenging for these samples, the magnitude of the difference is

CONCLUSIONS

The high mass transmission characteristics of a conjoined ion guide in the source region of a Q-IM-

TOF has been investigated

Excellent transmission is observed for species ranging from 150 kDa to 802 kDa with notable increases in

performance over a single source ion guide design

The optimum conjoined guide operating parameters

are broadly similar to those for lower molecular weight species allowing relatively generic settings

Further studies are needed to qualify transmission differences between the single and conjoined guides

Figure 2 Diagrams of the conjoined stacked ring ion guides.

Opposite phases

of RF voltage ap-plied to adjacent

electrodes

Conjoined Ion Guides

Electrodes

~120mm

15mm

5mm

11mm

Ions + GasFrom APISource

Rough Pump

Gas

Ions

DifferentialAperture

Conjoined Ion Guides

Ion Trajectories

Figure 3 Schematic diagrams illustrating the operating principle of the conjoined ion guide.

X

Y Diffuse Ion Cloud

Compact Ion Cloud

Elec

tric

Fie

ld

PotentialContours

X

Y

m/z7500 10000

%

0

100

4mb 5mb 6mb 7mb 8mb 9mb 10mb

Conjoined Ion Guide Pressure

Inte

nsi

ty

Figure 9 NanoESI mass spectra of GDH as a function of con-

joined ion guide pressure.

Figure 4 NanoESI mass spectra of MAB: Synapt G2-S.

24+ charge state peak FWHM = 28 m/z, ~350 counts/sec peak top

m/z5000 5250 5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 8250 8500 8750

%

0

100

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%

0

100

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%

0

100

24+ 24+

ADC60 secs

ADC1 sec

TDC60 secs

TDC1 sec

Figure 5 NanoESI mass spectra of GDH: Synapt G2-S.

38+ charge state peak FWHM = 53 m/z, ~170 counts/sec peak top

m/z7000 7500 8000 8500 9000 9500 10000 10500

%

0

100

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%

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38+ 38+

ADC60 secs

ADC1 sec

TDC60 secs

TDC1 sec

Figure 6 NanoESI mass spectra of GroEL: Synapt G2-S.

67+ charge state peak FWHM = 30 m/z, ~70 counts/sec peak top

m/z10000 10500 11000 11500 12000 12500 13000 13500

%

0

100

m/z10000 10500 11000 11500 12000 12500 13000 13500

%

0

100

m/z10000 10500 11000 11500 12000 12500 13000 13500

%

0

100

m/z10000 10500 11000 11500 12000 12500 13000 13500

%

0

10067+ 67+

ADC60 secs

ADC1 sec

TDC60 secs

TDC1 sec

Figure 8 Transmission plots through the conjoined ion guide as a

function of DC offset voltage (at 1 MHz and 320 V pk-pk RF).

0

20

40

60

80

100

0 20 40 60 80

No

rmal

ised

Tra

nsm

issi

on (%

)

DC Offset (V)

MAB

GDH

GroEL

m/z 455

Figure 7 Transmission plots through the conjoined ion guide as a func-

tion of RF pk-pk voltage (at 1 MHz and 25V DC offset).

0

20

40

60

80

100

0 50 100 150 200 250 300 350

No

rmal

ised

Tra

nsm

issi

on

(%)

RF Voltage (V)

MAB

GDH

GroEL

m/z 455

m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

%

0

100

m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

%

0

100x25

G2

G2-S

Figure 10 ESI mass spectra of GFP fragment ions obtained using

the Synapt G2 and Synapt G2-S systems.

being investigated further. Figure 12 highlights the data

quality obtainable using the Synapt G2-S system with the

improved transmission of the conjoined guide.

Figure 12 MS/MS spectra obtained using the Synapt G2-S sys-

tem. With and without quadrupole mass isolation.

m/z2500 5000 7500 10000 12500 15000 17500 20000 22500 25000 27500 30000

%

0

100

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%

0

10011801

1735

20701

x1011630

1790

20700

20137

21291

21915

22585

Collision Voltage = 75 V

x10

Figure 11 NanoESI mass spectra obtained using the Synapt G2

and G2-S systems.

m/z10000 10500 11000 11500 12000 12500 13000 13500

%

0

100

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100x20

x20

G2

G2-S

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G2-S

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G2-S