Mass Spectrometry Histochemistry of Human FFPE Material Getting Ready for the Clinic Peter Verhaert (1); Kenneth Verheggen (1); Nivedita Bhattacharya (1); Remco Crefcoeur (1); Samruddhi Chawan (1,2), Yury Tsybin (3); Konstantin Nagornov (3); Gilles Frache (4); Cecilia Lindskog-Bergström (5); Emma Lundberg (5); Per Andrén (6); Marthe Verhaert (1,2,7); Sandrine Aspeslagh (2); Raf Sciot (7); Kris Van Der Steen (8); Lin Zhang (9); Ling Shan (9); Dick Swaab (9); Wim Develter (10); Marc Ramael (10) (1) ProteoFormiX, Beerse, Belgium (2) University Hospital, Jette (Brussels), Belgium (3) Spectroswiss, Lausanne, Switzerland (4) Luxembourg Institute of Science and Technology, Esch-sur-Alzette, Luxembourg (5) Human Protein Atlas, Uppsala & Stockholm, Sweden (6) Science for Life Labs, Uppsala Sweden (7) University Hospital, Leuven, Belgium (8) Onze-Lieve-Vrouw Ziekenhuis, Aalst, Belgium (9) The Netherlands Institute for Neuroscience, Amsterdam, the Netherlands (10) Heilig-Hart Ziekenhuis, Lier, Belgium Peter Verhaert (Presenter) ProteoFormiX

Introduction
In our research facility at [email protected], we run a platform to discover candidate biomarker molecules for diseases with a high medical need. Our analytical workflow uses histological slides of formalin-fixed paraffin-embedded (FFPE) tissue straight from the clinic. Biomarker molecules are identified, with simultaneous mapping of their location on the tissue section. In analogy with immunohistochemistry, this imaging method is designated mass spectrometry histochemistry (MSHC). With MSHC we explore the vast collections of well-documented human neuronal tissues conserved in world famous tissue banks, as well as in modern hospital pathology archives.
By mapping all FFPE detectable biomolecules (particularly peptides, neurotransmitters and metabolites), we are compiling the FFPE Biomolecular Atlas of the Human Body.

Methods
Next to the large availability of FFPE samples and their extraordinary stability, a major advantage of MSHC is that it is a completely untargeted technique which directly links all existing histopathology knowledge with novel biomolecular information.
We have modified and fitted a high-resolution MALDI source (MassTech AP MALDI UHRTM) with a high-resolution FTMS analyzer (ThermoFisher Scientific LTQ Orbitrap VelosTM).
MSHC performance is benchmarked employing sections from biobanked human 'model' tissues.
Tissue blocks representing relevant Homo sapiens diseases are sectioned on a regular microtome at 5 µm thickness and deposited on regular microscope glass slides.
Slides are coated with a spray of DHB using an automated spraying device (HTX TM5TM Sprayer). Multi-Gb MSHC data files are processed, analyzed, and visualized using MozaicTM software (Spectroswiss).

Preliminary Data
MSHC allows routine imaging of biomolecules, including neuropeptides as well as biogenic amines and metabolites with high mass accuracy and 20 µm lateral resolution (between 5 and 10 µm can be achieved).
Data of reference secretory peptides (e.g., the disulfide bridge-linked neuropeptides vasopressin and oxytocin in pituitary/hypothalamus), establish MSHC as single cell 'multi-omics' technology. Multi-Gb MSHC data sets from biopsy as well as autopsy samples of patients exhibiting various stages histologically diagnosed diseases with high unmet needs are currently explored with high priority.
We warmly welcome interested collaborators to our Homo sapiens FFPE Biomolecular Body Atlas project.

Novel Aspect
Biomolecule mapping of secretory peptides and metabolites on histological sections of human FFPE clinical tissue representing health and disease.