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.