Advancing Mass Spectrometry Imaging: Immuno-DESI-MSI Unveils the Spatial Proteomic Landscape

Desorption electrospray ionization (DESI) is an ambient ionization technique that can be coupled to mass spectrometry imaging (MSI) for chemical analysis of samples at atmospheric conditions. DESI employs a fast-moving charged solvent spray, at an angle relative to the sample surface, to extract analytes from the surfaces and propel the secondary ions toward the mass analyzer. This tandem technique can be used to analyze a wide range of small molecules including metabolites, lipids, synthetic drugs, natural chemical products, and illegal dopants across the test sample surface. Therefore, DESI-MSI has been applied in a wide variety of sectors including food and drug administration, environmental monitoring agencies, clinical, pharmaceutical and biotechnology industries. However, a significant challenge has persisted in extending DESI-MSI to larger macromolecules, particularly proteins. The ultrahigh molecular weight, poor ionization efficiency, and low abundance of proteins make their direct analysis and imaging a formidable task. Researchers have made commendable efforts to tackle this issue through various approaches, such as ammonium bicarbonate addition, spray desorption collection-based off-line analysis, native DESI, nano-DESI, and DESI combined with FAIMS (Field Asymmetric Ion Mobility Spectrometry). While these strategies have made progress, a versatile and comprehensive solution for imaging all types of proteins without molecular weight constraints remains elusive.

In a new study published in Angewandte Chemie International Edition by Dr. Xiaowei Song,  and Richard N. Zare and their collabators Dr. Chao Li, Dr. Tianhao Zhou, and Dr. Qingce Zang developed a new immuno-DESI-MSI cutting-edge technique at Stanford University that overcomes existing limitations and provides a new perspective on the spatial distribution of functional macromolecules within biological tissues.

The new method is founded on the principle of using labeled molecules, cross-linked to bio-specific antibodies, to report the position and quantity of target macromolecules. Unlike traditional optical signal-based imaging, such as immunofluorescent microscopy (IFM), which is limited by signal overlap, mass spectrometry-based immunoassays can process thousands of ion channels simultaneously, providing a comprehensive molecular phenotype.

The authors developed water microdroplet-cleavable mass tags. These mass tags, composed of boronic acid which can bind reversibly with cross-linker with a diol end. When sprayed onto tissue sections, the highly acidic microdroplets generated by DESI hydrolyze the boronate ester bond and release the mass tag from its tethered antibody. This method enables conventional DESI-MSI to map functional macromolecules across tissue sections, overcoming previous limitations regarding macromolecular imaging. Briefly, the research team designed and synthesized a probe consisting of a boronic acid mass tag (BMT), antibody (Ab), and cross-linker. The BMT is synthesized by reacting a fluorescent compound (ATTO series N-hydroxysuccinimide ester) with 3-aminophenylboronic acid. This bifunctional molecule serves both DESI-MSI and IFM purposes. The boron element has a special isotope pattern that make mass tags differentiable with those endogenous metabolites and lipids. The use of quaternary ammonium group guarantees the high ionization efficiency and sensitivity of mass tags.

Afterward, the antibody is conjugated with BMT through a cross-linker, creating a BMT-Ab complex that can specifically bind to the target macromolecular antigen. The BMT-Ab solution is incubated on tissue cryosections, allowing BMT-Ab to recognize and bind with the corresponding antigen. Then excess unbound BMT-Ab is removed by washing with an ammonium bicarbonate buffer solution. Next, the acidic water microdroplets sprayed during DESI-MSI hydrolyze the boronate ester bond, releasing free BMT molecules for desorption, ionization, and transport into the mass spectrometer. Finally, the authors collect the sequential DESI-MS and IFM images for further analysis.

It is noteworthy to mention that the authors provided compelling evidence of its effectiveness by targeting the epithelial growth factor receptor (EGFR) pathway, an important therapeutic target in cancer research. This pathway includes EGFR itself and several downstream signaling factors (Raf, ERK, p-MEK, p-Raf, p-ERK), and enzymes (PLC, PKC). The results indicate that immuno-DESI-MSI can successfully differentiate and quantify the spatial distribution of these macromolecules, providing a comprehensive view of the EGFR pathway’s activity within a tissue section. Furthermore, the authors findings showed the downstream metabolic changes induced by the anti-tumor drug lapatinib within the context of the EGFR pathway. It identifies alterations in energy metabolism, one-carbon unit metabolism, RNA synthesis, and redox homeostasis, shedding light on the multifaceted impact of the drug on tumor cells.

Overall, this new study expands our ability to conduct spatial multi-omics studies, which hold immense potential for biomarker-based disease diagnosis and unraveling molecular mechanisms underlying disease progression. Immuno-DESI-MSI’s ability to provide subcellular resolution, coupled with its quantitative capabilities, makes it a powerful tool for the fields of molecular biology, pharmacology, and clinical research.

In conclusion, the study led by Professor Richard Zare and his collaborators introduces a game-changing technique in the field of mass spectrometry imaging. Immuno-DESI-MSI, by leveraging water microdroplet-cleavable mass tags, offers an unprecedented view of the spatial distribution of functional macromolecules within biological tissues. The ability to visualize and quantify complex pathways and metabolic changes at a subcellular level has vast implications for our understanding of disease mechanisms and the development of targeted therapies. As this innovative method gains traction and evolves, it holds the potential to drive breakthroughs in various fields, including cancer research, drug development, and personalized medicine.