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Abstract No.: ThP-133
Session: Instrumentation and Methodologies for Imaging MS
Presentation date: Thu, Aug 31, 2006
Presentation time: 09:50 – 11:20

MALDI Imaging Mass Spectrometry of Neuropeptide Distributions in Brain Tissue Sections

Maarten A. F. Altelaar1, Robert P. J. de Lange2, Roger A. H. Adan2, Wolter J. Mooi3, Ron M. A. Heeren1, Sander R. Piersma1

1 FOM-Institute for Atomic and Molecular Physics, Amsterdam, Netherlands
2 Rudolf Magnus Institute of Neuroscience, Dept of Pharmacology and Anatomy, UMC, Utrecht, Netherlands
3 Department of Pathology, Free University Medical Centre, Amsterdam, Netherlands

Correspondence address: Maarten Altelaar, FOM-AMOLF, Biological Mass Spectrometry, Kruislaan 407, Amsterdam, 1098 SJ Netherlands.

Keywords: Drug; Imaging; MALDI; Neuropeptides.

Novel aspect: Imaging local neuropeptide acetylation and monitoring the influence of leptin on the activation and deactivation of the neuropeptides α-MSH and β-endorphin and the resulting effect on food intake regulation.

 

Introduction
Mass spectrometry offers great potential in imaging protein and peptide posttranslational modifications directly in tissue sections. Here, conventional techniques like fluorescence microscopy are hampered by the need for a molecule specific label. These labels often are not able to distinguish between the native and modified biomolecule.
The neuropeptides α-MSH and β-endorphin, derived from the precursor protein proopiomelanocortin (POMC), play an important role in the regulation of food intake in rats. Activation of α-MSH and deactivation of β-endorphin is controlled by posttranslational amino-terminal acetylation. Since both peptides are derived from the same precursor molecule this acetylation step is crucial in food intake regulation. Here, imaging mass spectrometry is used to study peptide acetylation directly in rat brain tissue sections as well as in human and rat pituitary tissue sections.

Method
Rats were decapitated without prior anesthesia and brains and pituitary glands were dissected and frozen in liquid isopentane, cooled to 50 °C on dry ice and then stored at 80 °C until sectioning. 10µm thick tissue sections were cut using a cryomicrotome. Sections were thaw-mounted on ITO-coated glass slides and were stored at 80 °C until use.

Prior to mass spectrometry tissue sections were brought to room temperature in a desiccator over a silica gel canister, briefly washed in cold 70% ethanol and dried at room temperature. Matrix (10 mg/ml 4-HCCA in 50% EtOH/0.1 % TFA) was deposited using a TLC sprayer followed by sputter coating of 5nm of gold.

MALDI stigmatic imaging MS was performed on an extensively modified Physical Electronics (Eden Prairie, MN) TRIFT-II (triple focusing ToF) mass spectrometer equipped with a wedge, diode pumped solid-state Nd-YAG laser source (BrightSolutions, Italy) and a phosphor screen/CCD camera combination.

Results
The ion optics of the mass spectrometric microscope have been designed such that a magnified (ion-optical) image of the surface is mapped onto a position sensitive detector. This allows us to perform stigmatic MALDI imaging directly on tissue sections. In the pituitary tissue of both rat and man, well-defined localization of the different peptides is observed in the distinct pituitary regions. Vasopressin, neurotensin and oxytocin can be found in the posterior pituitary, while des-, single and diacetylated α-MSH can be found in the intermediate pituitary.

In the rat brain localized acetylation of α-MSH can be shown along the third ventricle, which until now was not possible by immunohistochemistry. Two gradients of acetylated/desacetylated α-MSH were observed indicating activation and deactivation of the α-MSH signal. A comparative study between control and leptin treated rats is performed to reveal changes in acetylation of the α-MSH and β-endorphin peptides and the effect on the regulation of food intake.