Incorporation of FAIMS into LC-MS

‘omics’ analysis

Kayleigh L. Arthur, Matthew A. Turner, James C. Reynolds and Colin S. Creaser Centre for Analytical Science, Department of Chemistry, Loughborough University, Loughborough, LE11 3TU, U.K.

LC-MS

LC-FAIMS-MS

Chromatographic

Peak

Compensation

Field scan

– 11 CFs

Mass Spectra

6 s

1 s

Figure 4: Representation of LC-FAIMS-MS peak and

scan timescale and peak deconvolution for each CF

KAYLEIGH ARTHUR

Centre for Analytical Science, Department of Chemistry, Loughborough University

K.Arthur@lboro.ac.uk, uk.linkedin.com/kayleigh-arthur-0512348a

• In non-targeted ‘omics’ experiments such as metabolomics and proteomics,

typically liquid or gas chromatography (LC or GC) combined with mass

spectrometry (MS) is used to separate and analyse complex biological

matrices

• Molecular features can however be missed or remain hidden within the

dataset using these techniques due to:

o Trace level features unresolved from the noise

o Unresolved isomeric or isobaric species

• We propose the use of field asymmetric waveform ion mobility spectrometry

(FAIMS) in a scanning capacity (Figure 2) in conjunction with LC-MS to

improve peak capacity and reduce chemical noise

Agilent

6230

TOF MS

Drying gas flow

Spray shield

Miniaturised

FAIMS

Inlet capillary

Nebuliser Sheath

gas flow

Nozzle

Liquid flow

Figure 1: Schematic diagram

of the modified Agilent

JetStream electrospray

source with miniaturised

FAIMS device

• The data presented here used a miniaturised

FAIMS chip (ultraFAIMS, Owlstone Ltd.) located

in the modified ion source region of a time-of-

flight-MS (Agilent 6230 TOF), in front of the MS

inlet capillary (Figure 1)

• FAIMS is a gas phase ion

separation technique that

utilises an oscillating high

frequency (RF) waveform,

known as the dispersion

field (DF)

• Rapid separation of gas

phase ions is a result of

differences in an ion’s

mobility in a buffer gas

under alternating high and

low electric fields (Figure 2)

Figure 2: Representation of ion transmission through

planar FAIMS electrodes

• A small DC voltage known as the compensation field (CF) can be superimposed

on the DF in order to selectively transmit ions, or scan through CF values to

transmit ions • Separation based on ion mobility makes FAIMS

orthogonal to both LC and MS

• The main limitation to combining FAIMS with LC-

MS for scanning experiments is scanning the

FAIMS on a timescale compatible with a

chromatographic peak

Miniaturised chip-

based FAIMS in chip

housing 700 µm

100 µm

Side view of

FAIMS chip

Parallel pairs

of electrodes

To MS N2

Figure 3: Miniaturised FAIMS

device

• The fast scanning capabilities of

the miniaturised FAIMS chip

(Figure 3) allows an entire FAIMS

scan to be acquired per second,

allowing enough data points per

LC peak (Figure 4)

• Benefits from using LC-FAIMS-MS analysis compared to LC-MS:

o Trace level features can be separated from chromatographic noise

o Isomeric or isobaric species can be resolved at different CFs

o Improved peak capacity

• FAIMS conditions for use in LC-

FAIMS-MS analysis chosen from the

direct infusion of human urine into

FAIMS-MS (Figure 5)

• DF 240 Td, CF scanned between -0.9 –

4 Td at 0.49 Td step sizes

Figure 5: FAIMS-MS DF vs CF

plot of human urine infusion

Figure 6: LC-FAIMS-MS heat map of total

ion chromatograms at each CF

• From the deconvoluted total ion

chromatograms (Figure 6) can

already see multiple examples of

peaks at the same retention time

that appear in multiple FAIMS CFs,

but we can look closer at

individual m/z values (Figure 7)

• Ions at the same m/z and same retention

time can be separated using FAIMS

Figure 7: LC-FAIMS-MS heat map of

m/z 359.316

3 x10

0

1

2

3

Counts vs. Retention Time (min)

5.1 5.2 5.3

4 x10

0

1

2

3 5.212

3 x10

0

1

2

3 5.217

3 x10

0

0.5

1 5.203

Counts vs. Retention Time (min) 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6

LC-MS

FAIMS off

LC-FAIMS-MS CF 0.08 Td

LC-FAIMS-MS CF 2.04 Td

Figure 8: LC-FAIMS-MS chromatogram

comparison with LC-MS, of m/z 207.11 at

retention time 5.21 min

m/z 207.111

RT 5.21 min

FAIMS scanned

from CF -0.9 to 4 Td

LC-FAIMS-MS

FAIMS off = 1 peak, FAIMS on = 2 peaks

• Figure 8 shows an example of the

separation of isobaric species

(same m/z and RT) using LC-

FAIMS-MS and an increase in peak

capacity

• Figure 9 shows an example of the

reduction of chemical noise using

LC-FAIMS-MS so that the peak S:N

is improved using FAIMS

3 x10

0

2

4

3 x10

0

1

2

Counts vs. Retention Time (min)

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5

LC-MS FAIMS off

LC-FAIMS-MS

CF 0.08 Td

m/z 331.210, RT 4.24 min; FAIMS off = 0 peak, FAIMS on = 1 peaks

FAIMS off S:N = 2.7

FAIMS on S:N = 124.1

Figure 9: LC-FAIMS-MS chromatogram

comparison with LC-MS, of m/z 331.21 at

retention time 4.24 min

ACKNOWLEDGEMENTS

The authors thank Owlstone Ltd., Agilent Technologies and Loughborough University for financial and technical support

The authors thank the Royal Society of Chemistry for the award of a bursary for conference attendance