We report an atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) setup with a lateral resolution of 1.4 μm, a mass resolution greater than 100,000, and accuracy below ±2 p.p.m. We achieved this by coupling a focusing objective with a numerical aperture (NA) of 0.9 at 337 nm and a free working distance of 18 mm in coaxial geometry to an orbitrap mass spectrometer and optimizing the matrix application. We demonstrate improvement in image contrast, lateral resolution, and ion yield per unit area compared with a state-of-the-art commercial MSI source. We show that our setup can be used to detect metabolites, lipids, and small peptides, as well as to perform tandem MS experiments with 1.5-μm sampling areas. To showcase these capabilities, we identified subcellular lipid, metabolite, and peptide distributions that differentiate, for example, cilia and oral groove in Paramecium caudatum.

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We describe an atmospheric pressure matrix-assisted laser desorption-ionization mass spectrometry imaging system that uses long-distance laser triangulation on a micrometer scale to simultaneously obtain topographic and molecular information from 3D surfaces. We studied the topographic distribution of compounds on irregular 3D surfaces of plants and parasites, and we imaged nonplanar tissue sections with high lateral resolution, thereby eliminating height-related signal artifacts.

Metabolites, lipids, and other small molecules are key constituents of tissues supporting cellular programs in health and disease. Here, we present METASPACE, a community-populated knowledge base of spatial metabolomes from imaging mass spectrometry data. METASPACE is enabled by a high-performance engine for metabolite annotation in a confidence-controlled way that makes results comparable between experiments and laboratories. By sharing their results publicly, engine users continuously populate a knowledge base of annotated spatial metabolomes in tissues currently including over 3000 datasets from human cancer cohorts, whole-body sections of animal models, and various organs. The spatial metabolomes can be visualized, explored and shared using a web app as well as accessed programmatically for large-scale analysis. By using novel computational methods inspired by natural language processing, we illustrate that METASPACE provides molecular coverage beyond the capacity of any individual laboratory and opens avenues towards comprehensive metabolite atlases on the levels of tissues and organs.

The effect of double bond functionalisation for selective double bond localisation by ultraviolet photodissociation of phosphatidylcholines is investigated. Paternò-Büchi reactions in nanoESI emitter tips enable attachment of acetophenone to double bonds of unsaturated phosphatidylcholines after 100 s of 254 nm light irradiation with about 50-80% reaction yield. Functionalized phosphatidylcholines dissociate upon 266 nm irradiation yielding double bond selective fragment ions in contrast to results for ultraviolet photodissociation of unmodified lipids. Ultraviolet photodissociation of Paternò-Büchi modified lipids results in a selectivity increase of up to 2.2 towards double bond localisation compared collision-induced dissociation experiments. Double bond localisation is also possible with ultraviolet photodissociation when alkali metal ion attachment to Paternò-Büchi modified phosphatidylcholines occurs in contrast to classic collision-induced dissociation experiments. The developed methodology is used to differentiate lipid double bond isomers and applied to phosphatidylcholines from egg yolk to identify 15 phosphatidylcholines. Results from this study demonstrate that locally depositing energy in close vicinity to cleavable bonds via ultraviolet photodissociation can result in increased dissociation selectivity. This method can help to disentangle contributions from different structural elements in complex tandem mass spectra of lipids and aid to the structural characterization of phospholipids in a "top-down" approach.