Mass spectrometry imaging (MSI) is a powerful, label-free technique that provides detailed maps of hundreds of molecules in complex samples with high sensitivity and subcellular spatial resolution. Accurate quantification in MSI relies on a detailed understanding of matrix effects associated with the ionization process along with evaluation of the extraction efficiency and mass-dependent ion losses occurring in the analysis step. We present a critical summary of approaches developed for quantitative MSI of metabolites, lipids, and proteins in biological tissues and discuss their current and future applications. Expected final online publication date for the Annual Review of Physical Chemistry, Volume 72 is April 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Unraveling the complexity of the lipidome requires the development of novel approaches for the structural characterization of lipid species with isomer-level discrimination. Herein, we introduce an online photochemical approach for lipid isomer identification through selective derivatization of double bonds by reaction with singlet oxygen. Lipid hydroperoxide products are generated promptly after laser irradiation. Fragmentation of these species in a mass spectrometer produces diagnostic fragments revealing the C=C locations in the unreacted lipids. This approach uses an inexpensive light source and photosensitizer making it easy to incorporate into any lipidomics workflow. We demonstrate the utility of this approach for the shotgun profiling of C = C locations in different lipid classes present in tissue extracts using electrospray ionization (ESI) and ambient imaging of lipid species differing only by the location of C=C bonds using nanospray desorption electrospray ionization (nano-DESI).

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Mass spectrometry imaging (MSI) is a powerful tool for label-free mapping of the spatial distribution of proteins in biological tissues. We have previously demonstrated imaging of individual proteoforms in biological tissues using nanospray desorption electrospray ionization (nano-DESI), an ambient liquid extraction-based MSI technique. Nano-DESI MSI generates multiply charged protein ions, which is advantageous for their identification using top-down proteomics analysis. In this study, we demonstrate proteoform mapping in biological tissues with a spatial resolution down to 7 μm using nano-DESI MSI. A substantial decrease in protein signals observed in high-spatialresolution MSI makes these experiments challenging. We have enhanced the sensitivity of nano-DESI MSI experiments by optimizing the design of the capillary-based probe and the thickness of the tissue section. In addition, we demonstrate that oversampling may be used to further improve spatial resolution at little or no expense to sensitivity. These developments represent a new step in MSI-based spatial proteomics, which complements targeted imaging modalities widely used for studying biological systems.