For research use only. Not for use in diagnostic procedures.

Complimentary Use of MALDI FTICR-MS and TOF-SIMS Imaging: Approaches in an Invertebrate

ASMS 2013

Methods

Results

Manuel Liebeke1; Jens Fuchser2; Katherine A. Kellersberger3; Sarah Fearn4; David McPhail4; Jacob G. Bundy1

1Department of Surgery and Cancer, Imperial College London, London, UK, 2Bruker Daltonik GmbH, Bremen, Germany 3Bruker Daltonics Inc., Billerica, MA, USA 4Department of Materials, Imperial College London, London, UK

In a previous untargeted metabolomics study1, the metabolite 2-hexyl‐5‐ethyl-3-furansulfonic acid (HEFS, Figure 2) showed a toxicity induced reduction in earthworms. The results presented here focus on the specific localization of this metabolite in the invertebrate Lumbricus rubellus (widely distributed earthworm species) to receive a better understanding of its biological function. The application of Imaging Mass Spectrometry (IMS) techniques allows for whole-organism to sub-cellular metabolite localization. The use of MALDI in conjunction with an ultra-high resolution mass spectrometer with MS/MS capabilities makes in-situ identification of known and unknown metabolites feasible. In addition, TOF-SIMS with much higher spatial resolution but with a limited mass range gives information on detailed cellular distribution, making both methods complementary.

Conclusions •  HEFS located in earthworm intestine (2

independend MSI)

•  Highly accurate MS and MS/MS approaches with MALDI Imaging

•  Identified HEFS and unexpected HEFS related sulfonated compounds in the earthworm gut

•  Functional assays needed to identify biological role gut surfactant (bile acid equivalent?)

FTICR MALDI Imaging

Fig. 2. MS/MS Spectrum of 2-hexyl-5-ethyl-3-furan-sulfonic acid (HEFS) directly from tissue with ppb mass accuracy

Introduction

Fig. 3 Results of MALDI FTICR - and TOF SIMS - Imaging

The molecule of interest, 2-hexyl‐5‐ethyl-3-furansulfonic acid (HEFS) was identified and localized in earthworm intestine utilizing two different MSI technologies. High mass accuracy MALDI-FTMS yielded the localization at a 50µm spatial resolution. In addition, unexpected HEFS-related sulfonated compounds were identified in the earthworm gut by MS/MS approaches. An even more refined localization was enabled by the TOF-SIMS technology at a spatial resolution of 250nm and revealed a main HEFS concentration in the gut lumen and neighbouring cells where this class of compounds may be produced or stored.

The now established link between the toxicity-induced reduction of HEFS and the tissue of interest is key to designing biological assays which may unravel the biological role of HEFS and related sulfonated compounds. Early experiments to elucidate surface active properties of HEFS in conjunction with compound uptake through the earthworm gut revealed an interaction with several naturally occurring nutrients, demonstrating the importance of HEFS for earthworm metabolism.

.

We report here the combined use of Matrix Assisted Laser Desorption Ionization (MALDI) FTICR-MS to image tissue cross-sections (0.5cm2 – 50µm spatial resolution) and TOF-SIMS to further elucidate the cellular distribution of selected small molecules (125 – 500µm2 – 250 nm spatial resolution). Both IMS techniques revealed robust cross-platform tissue specific metabolite distributions. The MS/MS capabilities of the FTICR-MS were used to identify metabolites directly on the tissue section (Figure 2). LC-MSn separation of tissue extracts showed that co-localized mass spectral features represent individual metabolites with similar MS/MS characteristics. MALDI FTICR MS -homogeneous matrix (HCCA) deposition, with ImagePrep instrument (Bruker Daltonics) 12 Tesla FTICR MS (solariX, Bruker Daltonics) Selected mass range 130<m/z<1500 x-y-raster width of 50 µm using 1 KHz smartbeam™ II laser optics with a 30 µm laser focus TOF-SIMS -no matrix application needed -IONTOF ToF-SIMS5 instrument -Bi3+ ion beam in the burst alignment mode -charge compensation of the sample’s surface was supplied by an electron flood gun (20eV)

Fig.1 Relative amount of selected metabolites in L. rubellus sections and structure of the main metabolite in tissue extracts: 2-hexyl-5-ethyl-3-furansulfonic acid (HEFS). An adult worm was sectioned into 20 equal sized (~40mg) parts (longitudinal direction from head to tail). Metabolites quantified by 1H-NMR.

1000µm

Intensity of HEFS ion (259.1009Da) -metabolite confirmation by high mass accuracy and MS/MS experiments on tissue section -50µm spatial resolution metabolite located within intestinal tissue

Intensity of HEFS fragment ion on a different cross section (taken next to earthworm clitellum) - ‘stitched’ image of ion maps -other ions show co-location with HEFS -no MS/MS capabilities (àMALDI-MS/MS) TOF-SIMS shows on ‘stitched’ image HEFS located in intestine tisue

Intensity of HEFS ion (purple) and tissue specific fatty acid ions (green) (overlay onto microscopic image) - After H&E staining confirmation of tissue types and organs -250nm spatial resolution high spatial res TOF-SIMS image proofs that gut lumen represents main HEFS location

1Bundy et al - Environmental Toxicology and Chemistry, Vol. 21, No. 9, pp. 1966–1972, 2002

Literature

172.991401-­‐

188.014881-­‐

244.077491-­‐

259.100951-­‐

-­‐MS2(qCID  259.10095)

140 160 180 200 220 240 m/z0

1

2

3

8x10Intens.

Regenwurm_neg_259_QCID_0_E8_000003.d: -MS2(qCID 259.10095)

[M-H-CH3]- [M-H-C5H11]-

[M-H-C6H14]-