Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2015 Waters Corporation
INTRODUCTION
'Steroidomics' is the qualitative and quantitative study of steroid-type molecules found within the metabolome. Bile acids for example, are classified as acidic sterols that are synthesised mainly by the liver from cholesterol and aid digestion and fat solubilisation. The presence of
multiple isomeric bile acids poses a great challenge for steroidomic research. Ion mobility -mass spectrometry (IM-MS) was combined with molecular modelling for the separation and configurational analysis of thirteen medically relevant bile acids. The usefulness of the rotationally averaged collision cross-section (CCS) information derived from the experiment-tally derived IM measurements of relevant bile acids may be used to enhance specificity and augment steroidomic-type research and aid the diagnosis, prognosis and management of disease.
METHODS
Mass Spectrometry
MS: Vion IMS Q-ToF and Synapt G2-Si Mode: ESI and MALDI (-VE)
Capillary voltage: 2kV Cone: 40V
Source temperature: 110°C Scan rate: 1 spectrum/s
ESI
The bile acids were infused at a concentration of 0.1ng/µL (MeOH) and the signal attenuated with the DRE lens
MALDI-Imaging
Isomeric bile acid mixtures were spotted on a 30 µm mouse
brain section mounted on a glass slide. The slide was spray coated with matrix using the SunChrom SunCollect Sprayer.
30 coats were applied at a flow rate of 20 µL/min. MALDI
images were processed using High Definition Imaging software v1.3. Matrix: 9-aminoacridine (0.5 mg/mL in 4:1 EtOH:H2O).
Ion Mobility
Mobility bath gas: N2 (Vion) N2 (G2-Si) CO2 (G2-Si only)
Ion mobility cell: ~3.0mbar ~3.0mbar ~3.0mbar
IMS Wave velocity: 850 m/s 900 m/s 900 m/s Trap Wave Height: 40-60V 40V 40V
Workflow
ESI-MS was used to measure ion drift-times upon a hybrid ion
mobility/ quadrupole / oa-ToF MS (Vion IMS Q-ToF), ESI and MALDI-imaging was also used upon a hybrid quadrupole / ion
mobility / oa-ToF MS (Synapt G2-Si). N2 was used as the
mobility gas in both instruments and CO2 in the Synapt G2-Si only.
THE ANALYSIS OF BILE ACIDS: ENHANCEMENT OF SPECIFICITY USING AN ION MOBILITY-TOFMS BASED APPROACH
Jonathan P Williams1, Martin Palmer1, Jonas Abdel-Khalik2, Yuqin Wang2, Sarah M Stow3, Mark Towers1, Giuseppe Astarita1, James Langridge1 and William J Griffiths2 1 Waters Corporation, Wilmslow, Manchester UK; 2 College of Medicine, Swansea University UK; 3 Laboratory for Structural Mass Spectrometry, Vanderbilt University, TN, USA
RESULTS
New and improved methods were sought for the identification, quantification, and characterization of bile acids, oxysterols, and other sterols and steroids. The involvement of these molecules in
neurogenesis and immunity is investigated. The use of IM as an analytical tool to aid direct infusion ESI, DESI and
MALDI shotgun steroidomic-type analysis was investigated. Bile acids present themselves in biological type samples as complex mixtures. Structural information may be obtained using MS/MS but in the absence
of a chromatographic step, unambiguous characterisation using MS/MS can be challenging since the selected precursor ion can be composed of chemical isomers and interfering isobaric ions.
The individual rotationally averaged CCS measured experimentally using direct infusion ESI-T-Wave ion mobility are shown in Table 1. The results
represent an average of three measurements. Isomeric bile acids are highlighted in grey.
CONCLUSIONS
Good correlation was achieved between the two T-
Wave ion mobility instruments used in this study.
In combination with accurate mass measurement,
the additional molecular descriptor of CCS can aid bile
acid ion identification
The results indicate that the addition of CCS
measurements to searchable databases within a ‘steroidomic-type’ workflow increases the specificity
and selectivity of bile acid analysis, improving the confidence in identification compared to traditional
analytical approaches
Structures of the bile acids.
IMS comprises a travelling wave RF ion guide, which incorpo-
rates a repeating sequence of transient DC pulses to propel ions through the guide in the presence of N2 bath gas. Upon
exiting the IMS cell, ions can be selected with the quadrupole and undergo CID for structural elucidation prior to detection
with the TOFMS. The T-Wave mobility device was calibrated for estimated rotationally averaged TWCCSN2 measurements using
drift tube obtained DTCCSN2 measurements of ions produced from polyalanine.
Modeling and Theoretical CCS determination
Obtain structure from PubChem Remove hydrogen to create deprotonated molecules [M-H]-
of the bile acids Run Gaussian Optimisation for starting structure and partial
charges Run Distance Geometry to generate a set of conformations.
8000 conformation limit was set for the deprotonated mole-cules of the bile acids
Run energy minimization for candidate low energy confor-mations
Theoretical CCS were obtained using the Trajectory Method in MOBCAL and from preliminary N2 parameters using the
Projection Superposition Approximation method
Fig. 1 Schematic of the Vion IMS Q-ToF
Fig.1 shows a schematic of Vion. In brief, the instrument com-
prises an IM separation device, a quadrupole and segmented collision cell prior to the TOFMS. Ions are accumulated in the
trap travelling-wave (T-Wave) and periodically released into the T-Wave IM where they separate according to their mobil-
ity.
Direct infusion-ESI Ion Mobility MS: CCS measurements
of the bile acids investigated
Fig. 2 Overlaid drift times (ms) of DCA, CA, CDA and HA. UA has a
very similar drift time to CDA and is different by only 1 scan (the flight time for the pusher frequency). The inset shows an overlay of
the individual drift times (ms) for UA, CDA and HA using a higher T-Wave ion mobility velocity of 1500 m/s.
Time9.00 10.00
%
0
100
Time1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
%
0
100
1. DCA 2. CA
3. CDA 4. HA
UA
CDA
HA
1
2 3
4
MALDI Imaging Ion Mobility MS measurements of the
isomeric bile acids deoxycholic acid and hyodeoxy-cholic acid
Fig. 3 MALDI-Imaging Synapt G2-Si of DCA and HA. Although
isomeric, the two bile acids, previously been detected in brain. could easily be differentiated using ion mobility and detected
where spotted on to the mouse brain tissue section.
Gas-Phase separation re-optimisation of bile acid isomers;
effect of mobility gas alteration from N2 to CO2
CDA
Time2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50
%
0
100
2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50
%
0
100
2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50
%
0
100
DCA
DCA
DCA
UA
HA
Fig. 4 Gas-phase separation optimisation for the bile acid iso-
mers obtained in N2 upon the Synapt G2-Si.
Time2.00 2.50 3.00 3.50 4.00 4.50 5.00
%
0
100
2.00 2.50 3.00 3.50 4.00 4.50 5.00
%
0
100
2.00 2.50 3.00 3.50 4.00 4.50 5.00
%
0
100
DCA HA
UA
DCA
DCA
CDA
Fig. 5 Gas-phase separation optimization of the bile acid iso-
mers obtained in CO2 upon the Synapt G2-Si
Modeling and Theoretical CCS generation
Fig. 6 Experimental CCS vs. theoretical CCS ranges for CCS
values obtained in N2 drift gas. Theoretical values were ob-tained using the Trajectory Method (green) in MOBCAL and
from the PSA Method (blue). Theoretical conformations were generated with distance geometry.
Fig. 7 Sample conformations for the bile acids obtained from
computational modeling for a) Glycodeoxycholic Acid, b) Taurodeoxycholic Acid, c) Deoxycholic Acid, and d) Chenode-
oxycholic Acid. Bile acids that have more interactions between the carboxylic or sulfonate end group on the tail structure with
the hydroxyl groups on the fused ring system correspond to the bile acids that fall closer to the lower bound on the theo-
retical range.
BILE ACID [M-H]- VionCCSN2 (Å2) G2-SiCCSN2
(Å2)
DEOXYCHOLIC ACID 202.6 198.9
CHOLIC ACID 204.1 200.8
CHENODEOXYCHOLIC ACID 209.0 205.5
LITHOCHOLIC ACID 208.6 204.6
URSODEOXYCHOLIC ACID 208.3 205.5
GLYCODEOXYCHOLIC ACID 199.8 196.9
GLYCOCHOLIC ACID 202.7 200.0
HYODEOXYCHOLIC ACID 209.6 206.5
TAUROCHENODEOXYCHOLIC ACID 208.4 204.9
GLYCOCHENODEOXYCHOLIC ACID 201.9 198.0
TAUROLITHOCHOLIC ACID 208.1 204.1
TAURODEOXYCHOLIC ACID 207.0 203.9
TAUROCHOLIC ACID 208.4 205.7
Table 1 VionCCSN2 and G2-SiCCSN2 of [M-H]- of the Bile Acids investigated
Representative conformations from distance geometry
modeling for the bile acids investigated