232
ionization imaging MS (MALDI IMS) is a technology that allows the molecular
mapping of potentially thousands of analytes from a tissue’s surface [3]. Spatial
molecular studies leveraging MALDI IMS during C. difficile infections will provide
insight into both the mechanisms and effects of colonization on the gastrointestinal
environment.
19.2 Methods
C57BL/6 mice were orally infected with C. difficile to an endpoint at three days.
Mice were sacrificed and intestines were harvested and wrapped in a spiral manner,
termed a ‘swissroll’, followed by freezing on liquid nitrogen. Intestines were sectioned at 10 μm thickness and thaw mounted onto conductive ITO coated slides or
vinyl slides. Fluorescence microscopy images were acquired prior to preparation for
IMS analysis. Samples were washed to remove salts and/or lipids and coated with
(E)-4-(2,5-dihydroxyphenyl)but-3-en-2-one (2,5-cDHA) using an aerosol sprayer.
High spatial resolution lipid and small molecule images were acquired using a prototype MALDI timsTOF Pro MS. Protein images were acquired using 15 T FT-ICR
MS. Post-processing included a second collection of fluorescence microscopy
images for accurate registration of data from different modalities.
19.3 Preliminary Data
MALDI MS, laser ablation inductively-coupled plasma MS, fluorescent, and bright
field microscopy imaging data have been generated from a control mouse as proof
of principle experiments. LA-ICP IMS data shows distinctions in localization of key
nutrient metals and elements between the luminal content and tissue in control
intestine samples. IMS data of lipids and proteins from the small intestine contain
species that clearly localize to either the host epithelium or the luminal contents.
Specifically, signals at m/z 1549.93 and m/z 1522.41 from positive ionization mode
data localize to the luminal contents, while peaks at m/z 810.55 and m/z 895.73
localize to the epithelium. C. difficile infected cecum samples also showed strong
epithelial localization at m/z 885.57 and m/z 936.62. However, lipids at m/z 741.54
were observed to localize primarily at the distal end. The proof-of-concept lipid
imaging experiments were collected using a prototype MALDI timsTOF platform
operated in Q-TOF mode. This platform allowed for rapid collection (20 pixels/s) of
high spatial resolution ion images (10 μm). MALDI FT-ICR protein images showed
similar trends to lipid images within localization substructures. Signals at m/z
4279.08 and m/z 8565.11 both show localization to the epithelium of the host.
Ubiquitin, with a signal at m/z 8565.11, was also identified to localize exclusively to
the epithelium. Higher-mass proteins, however, generally localized only to the
luminal contents. Additionally, taurocholic acid (m/z 514.29), a primary bile acid
E. R. Guiberson et al.
ionization imaging MS (MALDI IMS) is a technology that allows the molecular
mapping of potentially thousands of analytes from a tissue’s surface [3]. Spatial
molecular studies leveraging MALDI IMS during C. difficile infections will provide
insight into both the mechanisms and effects of colonization on the gastrointestinal
environment.
19.2 Methods
C57BL/6 mice were orally infected with C. difficile to an endpoint at three days.
Mice were sacrificed and intestines were harvested and wrapped in a spiral manner,
termed a ‘swissroll’, followed by freezing on liquid nitrogen. Intestines were sectioned at 10 μm thickness and thaw mounted onto conductive ITO coated slides or
vinyl slides. Fluorescence microscopy images were acquired prior to preparation for
IMS analysis. Samples were washed to remove salts and/or lipids and coated with
(E)-4-(2,5-dihydroxyphenyl)but-3-en-2-one (2,5-cDHA) using an aerosol sprayer.
High spatial resolution lipid and small molecule images were acquired using a prototype MALDI timsTOF Pro MS. Protein images were acquired using 15 T FT-ICR
MS. Post-processing included a second collection of fluorescence microscopy
images for accurate registration of data from different modalities.
19.3 Preliminary Data
MALDI MS, laser ablation inductively-coupled plasma MS, fluorescent, and bright
field microscopy imaging data have been generated from a control mouse as proof
of principle experiments. LA-ICP IMS data shows distinctions in localization of key
nutrient metals and elements between the luminal content and tissue in control
intestine samples. IMS data of lipids and proteins from the small intestine contain
species that clearly localize to either the host epithelium or the luminal contents.
Specifically, signals at m/z 1549.93 and m/z 1522.41 from positive ionization mode
data localize to the luminal contents, while peaks at m/z 810.55 and m/z 895.73
localize to the epithelium. C. difficile infected cecum samples also showed strong
epithelial localization at m/z 885.57 and m/z 936.62. However, lipids at m/z 741.54
were observed to localize primarily at the distal end. The proof-of-concept lipid
imaging experiments were collected using a prototype MALDI timsTOF platform
operated in Q-TOF mode. This platform allowed for rapid collection (20 pixels/s) of
high spatial resolution ion images (10 μm). MALDI FT-ICR protein images showed
similar trends to lipid images within localization substructures. Signals at m/z
4279.08 and m/z 8565.11 both show localization to the epithelium of the host.
Ubiquitin, with a signal at m/z 8565.11, was also identified to localize exclusively to
the epithelium. Higher-mass proteins, however, generally localized only to the
luminal contents. Additionally, taurocholic acid (m/z 514.29), a primary bile acid
E. R. Guiberson et al.