High-throughput single-cell mass spectrometry enables metabolic network analysis by resolving phospholipid C[double bond, length as m-dash]C isomers.

Single-cell mass spectrometry (MS) is an essential technology for sensitive and multiplexed analysis of metabolites and lipids for cell phenotyping and pathway studies. However, the structural elucidation of lipids from single cells remains a challenge, especially in the high-throughput scenario. Technically, there is a contradiction between the inadequate sample amount (i.e. a single cell, 0.5-20 pL) for replicate or multiple analysis, on the one hand, and the high metabolite coverage and multidimensional structure analysis that needs to be performed for each single cell, on the other hand. Here, we have developed a high-throughput single-cell MS platform that can perform both lipid profiling and lipid carbon-carbon double bond (C[double bond, length as m-dash]C) location isomer resolution analysis, aided by C[double bond, length as m-dash]C activation in unsaturated lipids by the Paternò-Büchi (PB) reaction and tandem MS, termed single-cell structural lipidomics analysis. The method can achieve a single-cell analysis throughput of 51 cells per minute. A total of 145 lipids were structurally characterized at the subclass level, of which the relative abundance of 17 isomeric lipids differing in the location of C[double bond, length as m-dash]C from 5 lipid precursors was determined. While cell-to-cell variations in MS1-based lipid profiling can be large, an advantage of quantifying lipid C[double bond, length as m-dash]C location isomers is the significantly improved quantitation accuracy. For example, the relative standard deviations (RSDs) of the relative amounts of PC 34:1 C[double bond, length as m-dash]C position isomers in MDA-MB-468 cells are half smaller than those measured for PC 34:1 as a whole by MS1 abundance profiling. Taken together, the developed method can be effectively used for in-depth structural lipid metabolism network analysis by high-throughput analysis of 142 MDA-MB-468 human breast cancer cells.

Rapid detection of IDH mutations in gliomas by intraoperative mass spectrometry.

The development and performance of two mass spectrometry (MS) workflows for the intraoperative diagnosis of isocitrate dehydrogenase (IDH) mutations in glioma is implemented by independent teams at Mayo Clinic, Jacksonville, and Huashan Hospital, Shanghai. The infiltrative nature of gliomas makes rapid diagnosis necessary to guide the extent of surgical resection of central nervous system (CNS) tumors. The combination of tissue biopsy and MS analysis used here satisfies this requirement. The key feature of both described methods is the use of tandem MS to measure the oncometabolite 2-hydroxyglutarate (2HG) relative to endogenous glutamate (Glu) to characterize the presence of mutant tumor. The experiments i) provide IDH mutation status for individual patients and ii) demonstrate a strong correlation of 2HG signals with tumor infiltration. The measured ratio of 2HG to Glu correlates with IDH-mutant (IDH-mut) glioma (P < 0.0001) in the tumor core data of both teams. Despite using different ionization methods and different mass spectrometers, comparable performance in determining IDH mutations from core tumor biopsies was achieved with sensitivities, specificities, and accuracies all at 100%. None of the 31 patients at Mayo Clinic or the 74 patients at Huashan Hospital were misclassified when analyzing tumor core biopsies. Robustness of the methodology was evaluated by postoperative re-examination of samples. Both teams noted the presence of high concentrations of 2HG at surgical margins, supporting future use of intraoperative MS to monitor for clean surgical margins. The power of MS diagnostics is shown in resolving contradictory clinical features, e.g., in distinguishing gliosis from IDH-mut glioma.

Accelerating protein aggregation and amyloid fibrillation for rapid inhibitor screening.

The accumulation and deposition of amyloid fibrils, also known as amyloidosis, in tissues and organs of patients has been found to be linked to numerous devastating neurodegenerative diseases. The aggregation of proteins to form amyloid fibrils, however, is a slow pathogenic process, and is a major issue for the evaluation of the effectiveness of inhibitors in new drug discovery and screening. Here, we used microdroplet reaction technology to accelerate the amyloid fibrillation process, monitored the process to shed light on the fundamental mechanism of amyloid self-assembly, and demonstrated the value of the technology in the rapid screening of potential inhibitor drugs. Proteins in microdroplets accelerated to form fibrils in milliseconds, enabling an entire cycle of inhibitor screening for Aß40 within 3 minutes. The technology would be of broad interest to drug discovery and therapeutic design to develop treatments for diseases associated with protein aggregation and fibrillation.

Electrospray-assisted cryo-EM sample preparation to mitigate interfacial effects.

Addressing interfacial effects during specimen preparation in cryogenic electron microscopy remains challenging. Here we introduce ESI-cryoPrep, a specimen preparation method based on electrospray ionization in native mass spectrometry, designed to alleviate issues associated with protein denaturation or preferred orientation induced by macromolecule adsorption at interfaces. Through fine-tuning spraying parameters, we optimized protein integrity preservation and achieved the desired ice thickness for analyzing target macromolecules. With ESI-cryoPrep, we prepared high-quality cryo-specimens of five proteins and obtained three-dimensional reconstructions at near-atomic resolution. Our findings demonstrate that ESI-cryoPrep effectively confines macromolecules within the middle of the thin layer of amorphous ice, facilitating the preparation of blotting-free vitreous samples. The protective mechanism, characterized by the uneven distribution of charged biomolecules of varying sizes within charged droplets, prevents the adsorption of target biomolecules at air-water or graphene-water interfaces, thereby avoiding structural damage to the protein particles or the introduction of dominant orientation issues.

Differentiating enantiomers by directional rotation of ions in a mass spectrometer.

Conventional mass spectrometry does not distinguish between enantiomers, or mirror-image isomers. Here we report a technique to break the chiral symmetry and to differentiate enantiomers by inducing directional rotation of chiral gas-phase ions. Dual alternating current excitations were applied to manipulate the motions of trapped ions, including the rotation around the center of mass and macro movement around the center of the trap. Differences in collision cross section were induced, which could be measured by ion cloud profiling at high resolutions above 10,000. High-field ion mobility and tandem mass spectrometry analyses of the enantiomers were combined and implemented by using a miniature ion trap mass spectrometer. The effectiveness of the developed method was demonstrated with a variety of organic compounds including amino acids, sugars, and several drug molecules, as well as a proof-of-principle ligand optimization study for asymmetric hydrogenation.

Risk Factors for Ocular Surface Irritation Symptoms in Inactive Mild and Moderate-to-Severe Graves' Orbitopathy.

INTRODUCTION: This study aims to analyze risk factors for ocular surface irritation symptoms in patients with non-corneal-damage inactive mild and moderate-to-severe Graves' orbitopathy (GO). METHODS: This retrospective study enrolled 307 patients with non-corneal-damage inactive GO admitted to Sun Yat-sen Memorial Hospital from April 2017 to September 2023. The activity and severity of GO were evaluated using the Clinical Activity Score (CAS) and the European Group on Graves' Orbitopathy (EUGOGO) classification, respectively. Multivariate logistic regression analysis was performed to analyze risk factors for ocular surface irritation symptoms. RESULTS: Among patients with inactive GO, for mild cases, CAS (P < 0.001), upper eyelid lag (P = 0.049), and extraocular muscle involvement (P = 0.019) in the symptomatic group were greater than those in the asymptomatic group, and multivariate logistic regression analysis demonstrated that upper eyelid lag (P = 0.048), CAS 1 (P < 0.001), CAS 2 (P = 0.005), and extraocular muscle involvement (P = 0.029) were risk factors for ocular surface irritation symptoms; for moderate-to-severe cases, CAS (P = 0.004), extraocular muscle involvement (P < 0.001), marginal reflex distance 1 (MRD1) (P = 0.030), and thyroid-stimulating hormone (TSH) (P = 0.034) in the symptomatic group were greater than those in the asymptomatic group, while multivariate logistic regression analysis indicated that extraocular muscle involvement (P = 0.018) and MRD1 (P = 0.012) were risk factors for ocular surface irritation symptoms. CONCLUSION: In non-corneal-damage inactive mild and moderate-to-severe GO, eyelid malposition and periocular muscle inflammation are risk factors for ocular surface irritation symptoms.

Graves' orbitopathy is the most common outward sign of Graves' disease. Patients with inactive Graves' orbitopathy often complain of ocular surface irritation symptoms. This study retrospectively collected clinical data from 307 patients with inactive Graves' orbitopathy and no concurrent corneal damage. The aim was to analyze risk factors for ocular surface irritation symptoms. Upper lid lag, eye movement disorder, and the Clinical Activity Score were found to be risk factors for mild cases. Eye movement disorder and the distance between the upper eyelid margin and corneal reflection point were risk factors for moderate-to-severe cases. To reduce symptoms, it may be helpful to treat inflammation around the eyes and address any eyelid abnormalities.

MS3 Imaging Enables the Simultaneous Analysis of Phospholipid C═C and sn-Position Isomers in Tissues.

Mass spectrometry (MS) imaging of lipids in tissues with high structure specificity is challenging in the effective fragmentation of position-selective structures and the sensitive detection of multiple lipid isomers. Herein, we develop an MS3 imaging method for the simultaneous analysis of phospholipid C═C and sn-position isomers by on-tissue photochemical derivatization, nanospray desorption electrospray ionization (nano-DESI), and a dual-linear ion trap MS system. A novel laser-based sensing probe is developed for the real-time adjustment of the probe-to-surface distance for nano-DESI. This method is validated in mouse brain and kidney sections, showing its capability of sensitive resolving and imaging of the fatty acyl chain composition, the sn-position, and the C═C location of phospholipids in an MS3 scan. MS3 imaging of phospholipids has shown the capability of differentiation of cancerous, fibrosis, and adjacent normal regions in liver cancer tissues.

Mobility-Modulated Sequential Dissociation Analysis Enables Structural Lipidomics in Mass Spectrometry Imaging.

Spatial lipidomics based on mass spectrometry imaging (MSI) is a powerful tool for fundamental biology studies and biomarker discovery. But the structure-resolving capability of MSI is limited because of the lack of multiplexed tandem mass spectrometry (MS/MS) method, primarily due to the small sample amount available from each pixel and the poor ion usage in MS/MS analysis. Here, we report a mobility-modulated sequential dissociation (MMSD) strategy for multiplex MS/MS imaging of distinct lipids from biological tissues. With ion mobility-enabled data-independent acquisition and automated spectrum deconvolution, MS/MS spectra of a large number of lipid species from each tissue pixel are acquired, at no expense of imaging speed. MMSD imaging is highlighted by MS/MS imaging of 24 structurally distinct lipids in the mouse brain and the revealing of the correlation of a structurally distinct phosphatidylethanolamine isomer (PE 18 : 1_18 : 1) from a human hepatocellular carcinoma (HCC) tissue. Mapping of structurally distinct lipid isomers is now enabled and spatial lipidomics becomes feasible for MSI.

Temperature-Tuning Desorption Ionization for Ambient Mass Spectrometry.

In the past decade, mass spectrometry (MS) has been widely used for a broad range of on-site applications. This is largely attributed to the rapid advancement of technologies, such as ambient ionization and mass spectrometer miniaturization. Here, we report the development of the temperature-tuning desorption ionization (TTDI) method for versatile on-site applications using a miniature MS system. A unique feature of TTDI is its dynamic temperature range applicable from 30 to 800 °C, which enables optimal desorption ionization applied for chemical and biological compounds through tuning the temperature at the sampling spot. The versatility of TTDI has been demonstrated through on-site MS analysis of a variety of samples, such as explosives on surfaces, drugs of abuse in biofluids, and screening biomarkers in tissues.

Efficient Sample Preparation System for Multi-Omics Analysis via Single Cell Mass Spectrometry.

Mass spectrometry (MS) has become a powerful tool for metabolome, lipidome, and proteome analyses. The efficient analysis of multi-omics in single cells, however, is still challenging in the manipulation of single cells and lack of in-fly cellular digestion and extraction approaches. Here, we present a streamlined strategy for highly efficient and automatic single-cell multi-omics analysis by MS. We developed a 10-pL-level microwell chip for housing individual single cells, whose proteins were found to be digested in 5 min, which is 144 times shorter than traditional bulk digestion. Besides, an automated picoliter extraction system was developed for sampling of metabolites, phospholipids, and proteins in tandem from the same single cell. Also, 2 min MS2 spectra were obtained from 700 pL solution of a single cell sample. In addition, 1391 proteins, phospholipids, and metabolites were detected from one single cell within 10 min. We further analyzed cells digested from cancer tissue samples, achieving up to 40% increase in cell classification accuracy using multi-omics analysis in comparison with single-omics analysis. This automated single-cell MS strategy is highly efficient in analyzing multi-omics information for investigation of cell heterogeneity and phenotyping for biomedical applications.

High-resolution separation of bioisomers using ion cloud profiling.

Elucidation of complex structures of biomolecules plays a key role in the field of chemistry and life sciences. In the past decade, ion mobility, by coupling with mass spectrometry, has become a unique tool for distinguishing isomers and isoforms of biomolecules. In this study, we develop a concept for performing ion mobility analysis using an ion trap, which enables isomer separation under ultra-high fields to achieve super high resolutions over 10,000. The potential of this technology has been demonstrated for analysis of isomers for biomolecules including disaccharides, phospholipids, and peptides with post-translational modifications.

Mass Spectrometry Imaging for Single-Cell or Subcellular Lipidomics: A Review of Recent Advancements and Future Development.

Mass Spectrometry Imaging (MSI) has emerged as a powerful imaging technique for the analysis of biological samples, providing valuable insights into the spatial distribution and structural characterization of lipids. The advancements in high-resolution MSI have made it an indispensable tool for single-cell or subcellular lipidomics. By preserving both intracellular and intercellular information, MSI enables a comprehensive analysis of lipidomics in individual cells and organelles. This enables researchers to delve deeper into the diversity of lipids within cells and to understand the role of lipids in shaping cell behavior. In this review, we aim to provide a comprehensive overview of recent advancements and future prospects of MSI for cellular/subcellular lipidomics. By keeping abreast of the cutting-edge studies in this field, we will continue to push the boundaries of the understanding of lipid metabolism and the impact of lipids on cellular behavior.

Metabolic Pathway of Monounsaturated Lipids Revealed by In-Depth Structural Lipidomics by Mass Spectrometry.

The study of lipid metabolism relies on the characterization of the lipidome, which is quite complex due to the structure variations of the lipid species. New analytical tools have been developed recently for characterizing fine structures of lipids, with C=C location identification as one of the major improvements. In this study, we studied the lipid metabolism reprograming by analyzing glycerol phospholipid compositions in breast cancer cell lines with structural specification extended to the C=C location level. Inhibition of the lipid desaturase, stearoyl-CoA desaturase 1, increased the proportion of n-10 isomers that are produced via an alternative fatty acid desaturase 2 pathway. However, there were different variations of the ratio of n-9/n-7 isomers in C18:1-containing glycerol phospholipids after stearoyl-CoA desaturase 1 inhibition, showing increased tendency in MCF-7 cells, MDA-MB-468 cells, and BT-474 cells, but decreased tendency in MDA-MB-231 cells. No consistent change of the ratio of n-9/n-7 isomers was observed in SK-BR-3 cells. This type of heterogeneity in reprogrammed lipid metabolism can be rationalized by considering both lipid desaturation and fatty acid oxidation, highlighting the critical roles of comprehensive lipid analysis in both fundamental and biomedical applications.

The role of altered lipid composition and distribution in liver fibrosis revealed by multimodal nonlinear optical microscopy.

Intracellular lipid accumulation is commonly seen in fibrotic livers, but its exact role in liver fibrosis remains elusive. Here, we established a multimodal nonlinear optical microscopy to quantitatively map distribution of biomolecules in fibrotic livers. Our data revealed that unsaturated triglycerides were predominantly accumulated in central vein area during liver fibrosis but not in portal vein area. Moreover, the lipid homeostasis was remarkably dysregulated in the late-stage compared to the early-stage fibrosis, including increased unsaturated triglycerides with decreased lipid unsaturation degree and decreased membrane fluidity. Such alterations were likely due to up-regulated lipogenesis, desaturation, and peroxidation, which consequently led to endoplasmic reticulum stress and cell death. Inspiringly, injured hepatocyte could be rescued by remodeling lipid homeostasis via either supply of unsaturated fatty acids or enhancement of membrane fluidity. Collectively, our study improves current understanding of the role of lipid homeostasis in fibrosis and open opportunities for treatment.

Tandem Mass Spectrometry Imaging Enables High Definition for Mapping Lipids in Tissues.

Mass spectrometry imaging (MSI) of lipids in biological tissues is useful for correlating molecular distribution with pathological results, which could provide useful information for both biological research and disease diagnosis. It is well understood that the lipidome could not be clearly deciphered without tandem mass spectrometry analysis, but this is challenging to achieve in MSI due to the limitation in sample amount at each image spot. Here we develop a multiplexed MS2 imaging (MS2 I) method that can provide MS2 images for 10 lipid species or more for each sampling spot, providing spatial structural lipidomic information. Coupling with on-tissue photochemical derivatization, imaging of 20 phospholipid C=C location isomers is also realized, showing enhanced molecular images with high definition in structure for mouse brain and human liver cancer tissue sections. Spatially mapped t-distributed stochastic neighbor embedding has also been adopted to visualize the tumor margin with enhancement by structural lipidomic information.

LipidOA: A Machine-Learning and Prior-Knowledge-Based Tool for Structural Annotation of Glycerophospholipids.

The Paternò-Büchi (PB) reaction is a carbon-carbon double bond (C═C)-specific derivatization reaction that can be used to pinpoint the location(s) of C═C(s) in unsaturated lipids and quantitate the location of isomers when coupled with tandem mass spectrometry (MS/MS). As the data of PB-MS/MS are increasingly generated, the establishment of a corresponding data analysis tool is highly needed. Herein, LipidOA, a machine-learning and prior-knowledge-based data analysis tool, is developed to analyze PB-MS/MS data generated by liquid chromatography-mass spectrometry workflows. LipidOA consists of four key functional modules to realize an annotation of glycerophospholipid (GPL) structures at the fatty acyl-specific C═C location level. These include (1) data preprocessing, (2) picking C═C diagnostic ions, (3) de novo annotation, and (4) result ranking. Importantly, in the result-ranking module, the reliability of structural annotation is sorted via the use of a machine learning classifier and comparison to the total fatty acid database generated from the same sample. LipidOA is trained and validated by four PB-MS/MS data sets acquired using different PB reagents on mass spectrometers of different resolutions and of different biological samples. Overall, LipidOA provides high precision (higher than 0.9) and a wide coverage for structural annotations of GPLs. These results demonstrate that LipidOA can be used as a robust and flexible tool for annotating PB-MS/MS data collected under different experimental conditions using different lipidomic workflows.

In-situ sampling of lipids in tissues using a porous membrane microprobe for direct mass spectrometry analysis.

Direct sampling of lipids from tissues for direct mass spectrometry (MS) analysis allows a quick profiling of lipidome, which is important for biomedical applications. In this work, we developed a polyporous polymeric membrane (PPM) microprobe for highly efficient sampling of lipids directly from tissue samples. The PPM was prepared by polypropylene with pores as large of 10 â€‹µm, facilitating the permeation of lipids from tissue surfaces. The PPM was coated onto a stainless steel wire with a thickness of ∼100 â€‹µm. The entire analysis procedure includes sampling of the lipids in tissue, washing the probe, and extraction spray ionization for MS analysis. The effectiveness was validated by analyzing mouse brain tissue samples. It showed high recoveries for a series of lipid classes in comparison with total lipid extraction method. Further demonstration was carried out with analysis of tissue samples from mouse liver, stomach, kidney and legs. With high physical strength and good chemical stability, the microprobe was also demonstrated for sampling lipids inside mouse kidney tissue samples. By incorporating a photochemical derivatization, a workflow was also developed for fast detection of lipid C[bond, double bond]C isomers in tissue samples. Finally, a microprobe array was also developed for simultaneous sampling of lipids from multiple sites on tissue surfaces.

Development of a Miniature Mass Spectrometry System for Point-of-Care Analysis of Lipid Isomers Based on Ozone-Induced Dissociation.

Disorder of lipid homeostasis is closely associated with a variety of diseases. Although mass spectrometry (MS) approaches have been well developed for the characterization of lipids, it still lacks an integrated and compact MS system that is capable of rapid and detailed lipid structural characterization and can be conveniently transferred into different laboratories. In this work, we describe a novel miniature MS system with the capability of both ozone-induced dissociation (OzID) and collision-induced dissociation (CID) for the assignment of sites of unsaturation and sn-positions in glycerolipids. A miniature ozone generator was developed, which can be operated at a relatively high pressure. By maintaining high-concentration ozone inside the linear ion trap, OzID efficiency was significantly improved for the identification of C═C locations in unsaturated lipids, with reaction times as short as 10 ms. Finally, the miniature OzID MS system was applied to the analysis of C═C locations and sn-positions of lipids from biological samples. Direct sampling and fast detection of changes in phospholipid isomers were demonstrated for the rapid discrimination of breast cancer tissue samples, showing the potential of the miniature OzID MS system for point-of-care analysis of lipid isomer biomarkers in complex samples.