Cellular lipidome is highly regulated through lipogenesis, rendering diverse double-bond positional isomers (C=C isomer) of a given unsaturated lipid species. In recent years, there are increasing reports indicating the physiological roles of C=C isomer compositions associated with diseases, while the biochemistry has not been fully understood due to the challenge in characterizing lipid isomers inherent to conventional mass spectrometry-based lipidomics. To address this challenge, we reported a universal, user-friendly, derivatization-based strategy, MELDI (mCPBA Epoxidation for Lipid Double-bond Identification), which enables both large-scale identification and spatial mapping of biological C=C isomers using commercial mass spectrometers without any instrument modification. With the developed liquid-chromatography mass spectrometry (LC-MS) lipidomics workflow, we elucidated more than 100 isomers among mono-and poly-unsaturated fatty acids and glycerophospholipids in both human serum, where novel isomers of low abundance were unambiguously quantified for the first time. The capability of MELDI-LC-MS in lipidome analysis was further demonstrated using the differentiated 3T3-L1 adipocytes, providing an insight into the cellular lipid reprogramming upon stearoyl-coenzyme A desaturase 1 (SCD1) inhibition. Finally, we highlighted the versatility of MELDI coupled with mass spectrometry imaging to spatially resolve cancer-associated alteration of lipid isomers in a metastatic mouse tissue section. Our results suggested that MELDI will contribute to current lipidomics pipelines with a deeper level of structural information, allowing us to investigate underlying lipid biochemistry.

Mass spectrometry imaging (MSI) using ambient ionization technique enables a direct chemical investigation of biological samples with minimal sample pretreatment. However, detailed morphological information of the sample is often lost due to its limited spatial resolution. In this study, predictive high-resolution molecular imaging was produced by the fusion of ambient ionization MSI with optical microscopy of routine hematoxylin and eosin (H&E) staining produces. Specifically, desorption electrospray ionization (DESI) and nanospray desorption electrospray ionization (nanoDESI) mass spectrometry are employed to visualize lipid and protein species on mice tissue sections. The resulting molecular

Paper spray ionization has been used as a fast sampling/ionization method for the direct mass spectrometric analysis of biological samples at ambient conditions. Here, we demonstrated that by utilizing paper spray ionization-mass spectrometry (PSI-MS) coupled with field asymmetric waveform ion mobility spectrometry (FAIMS), predictive metabolic and lipidomic profiles of routine breast core needle biopsies could be obtained effectively. By the combination of machine learning algorithms and pathological examination reports, we developed a classification model, which has an overall accuracy of 87.5% for an instantaneous differentiation between cancerous and noncancerous breast tissues utilizing metabolic and lipidomic profiles. Our results suggested that paper spray ionization-ion mobility spectrometry-mass spectrometry (PSI-IMS-MS) is a powerful approach for rapid breast cancer diagnosis based on altered metabolic and lipidomic profiles.

Biosynthesis of unsaturated lipids is highly regulated through cellular lipogenesis, rendering diverse double-bond positional isomers (C=C isomers) of a given unsaturated lipid species. However, identification and quantification of C=C isomers are usually challenging using conventional mass spectrometric analyses as they yield indistinguishable tandem mass spectra. To address this challenge, we proposed an easy-to-use, cost-effective, handy derivatization method, MELDI (mCPBA Epoxidation for Lipid Double-bond Identification), and its integrations with liquid chromatography mass spectrometry (LC-MS) lipidomics as well as desorption electrospray ionization (DESI) mass spectrometry imaging (MSI). Using MELDI-LC-MS, we identified more than 100 C=C isomers among mono-and poly-unsaturated fatty acids and glycerolphospholipids in human serum, where a variety of uncommon C=C isomers with low endogenous levels were quantified. The capability of MELDI-LC-MS in lipidome analysis was also demonstrated by the differentiated 3T3-L1 adipocytes, providing an insight into the cellular lipid reprogramming upon stearoylcoenzyme A desaturase 1 (SCD1) inhibition at the C=C isomer level. Additionally, combining MELDI with DESI-MSI allowed us to spatially resolve the tumor-associated change in C=C isomers in a metastatic mouse lung tissue section. Our results suggested that C=C isomers are potential indicators to disease-related alterations in cellular lipid homeostasis.

Nanospray desorption electrospray ionization mass spectrometry is an ambient ionization technique that is capable of mapping proteins in tissue sections. However, high-abundant molecules or isobaric interference in biological samples hampers its broad applications in probing low-abundant proteins. To address this challenge, herein we demonstrated an integrated module that coupled pneumatic-assisted nanospray desorption electrospray ionization mass spectrometry with high-field asymmetric ion mobility spectrometry. Using this module to analyze mouse brain sections, the protein coverage was significantly increased. This improvement allowed the mapping of low-abundant proteins in tissue sections with a 5 μm spatial resolution enabled by computationally assisted fusion with optical microscopic images. Moreover, the module was successfully applied to characterize melanoma in skin tissues based on the enhanced protein profiles. The results suggested that this integrating module will be potentially applied to discover novel proteins in cancers.

Rationale Natural products have been great sources for drug discovery. However, the structures of natural products are diverse and difficult to elucidate. Cordyceps militaris is a parasitic fungus which usually grows on host insects. The metabolites of C. militaris have been reported to act as chemotherapeutic agents. In this study, we aimed for the structural elucidation of specialized metabolites derived from C. militaris, and the metabolic impact in leukemia cells. Methods We describe a liquid chromatography data‐dependent mass spectrometric platform combining tandem mass analysis and molecular networking. Leukemia cells treated with C. militaris extract and control groups were visualized in terms of their metabolic profiles using Global Natural Product Social (GNPS) molecular networking. By this method, we were able to elucidate the structures of metabolites from medicinal fungus extracts and cancer cells and then to recognize their changes in a semi‐quantitative manner. Results Using C. militaris and leukemia cells as examples, we found that approximately 100 new ion species were present in the treated leukemia cells, suggesting a highly altered metabolic profile. Specifically, based on the tandem mass spectral similarity, we proposed that cordycepin, a key fungus‐derived therapeutic agent known for its antitumor activity, was transformed into its methylthio form in leukemia cells. Conclusions The platform described provides an ability to investigate complex molecular interactions of natural products in mammalian cells. By incorporating tandem mass spectrometry and molecular networking, we were able to reveal the chemical modification of crude bioactive compounds, for example potential bioactive compounds which might be modified from cordycepin. We envision that such a mass spectrometry (MS)‐based workflow, combined with other metabolomics platforms, would enable much wider applicability to cell biology and be of great potential to pharmacological study as well as drug discovery.

Adulteration of edible oils by the manufacturers has been found frequently in modern societies. Due to the complexity of the chemical contents in edible oils, it is challenging to quantitatively determine the extent of adulteration and prove the authenticity of edible oils. In this study, a robust and simple MALDI-TOF-MS platform for rapid fingerprinting of triacylglycerols (TAGs) in edible oils was developed, where spectral similarity analysis was performed to quantitatively reveal correlations among edible oils in the chemical level. Specifically, we proposed oil networking, a spectral similarity-based illustration, which enabled reliable classifications of tens of commercial edible oils from vegetable and animal origins. The strategy was superior to traditional multivariate statistics due to its high sensitivity in probing subtle changes in TAG profiles, as further demonstrated by the success in determination of the adulterated lard in a food fraud in Taiwan. Finally, we showed that the platform allowed quantitative assessment of the binary mixture of olive oil and canola oil, which is a common type of olive oil adulteration in the market. Overall, these results suggested a novel strategy for chemical fingerprint-based quality control and authentication of oils in the food industry.

Mass spectrometry imaging (MSI) is a powerful technique that reveals the spatial distribution of various molecules in biological samples, and it is widely used in pathology‐related research. In this review, we summarize common MSI techniques, including matrix‐assisted laser desorption/ionization and desorption electrospray ionization MSI, and their applications in pathological research, including disease diagnosis, microbiology, and drug discovery. We also describe the improvements of MSI, focusing on the accumulation of imaging data sets, expansion of chemical coverage, and identification of biological significant molecules, that have prompted the evolution of MSI to meet the requirements of pathology practices. Overall, this review details the applications and improvements of MSI techniques, demonstrating the potential of integrating MSI techniques into next‐generation pathology practices.