sample-preparation-for-single-cell-mass-spectrometry-metabolomics

Sample Preparation for Single Cell Mass Spectrometry Metabolomics Studies: Combined Cell Washing, Quenching, Drying, and Storage

Single-cell mass spectrometry (SCMS) has become an indispensable tool for studying cellular metabolism. Owing to advancements in modern mass spectrometry ...

single-cell-mass-spectrometry-imaging-using-desorption-electrospray

Single-Cell Mass Spectrometry Imaging Using Desorption Electrospray Ionization Coupled To Orbitrap Mass Spectrometers

Desorption electrospray ionization&#8211;mass spectrometry imaging (DESI&#8211;MSI) enables ambient, matrix-free molecular imaging with minimal sample ...

University of Oklahoma

University of Oklahoma Health Campus

Antibiotic resistance is a growing concern in the medical field. Drug-susceptible infections are often treated with β-lactam antibiotics, which bind to enzymes known as penicillin-binding proteins (PBPs). When the PBPs are disabled, the integrity of the cell wall is compromised, leading to cell lysis. Resistance renders β-lactam antibiotics ineffective, and clinicians turn to be more effective, but often more toxic, antibiotics. An alternative approach is combining antibiotics with compounds that disable resistance mechanisms. Previously, we have shown that low-molecular-weight 600 Da branched polyethylenimine restores β-lactam susceptibility to Gram-positive and Gram-negative pathogens with antibiotic resistance. In this study, this approach is extended to the homodimers of 600 Da BPEI that have improved potentiation properties compared to monomers of 600 Da BPEI and 1200 Da BPEI. The homodimers are synthesized by linking two 600 Da BPEI molecules with methylenebisacrylamide (MBAA). The resulting product was characterized with FTIR spectroscopy,  H NMR spectroscopy, checkerboard microbroth dilution assays, and cell toxicity assays. These data show that the 600 Da BPEI homodimer is more effective than 1200 Da BPEI toward the potentiation of oxacillin against methicillin-resistant Staphylococcus epidermidis and the potentiation of piperacillin against Pseudomonas aeruginosa.

Dimerization of 600 Da branched polyethylenimine improves β-lactam antibiotic potentiation against antibiotic-resistant Staphylococcus epidermidis and Pseudomonas aeruginosa.

Cell-cell interactions are critical for transmitting signals among cells and maintaining their normal functions from the single-cell level to tissues. In cancer studies, interactions between drug-resistant and drug-sensitive cells play an important role in the development of chemotherapy resistance of tumors. As metabolites directly reflect the cell status, metabolomics studies provide insight into cell-cell communication. Mass spectrometry (MS) is a powerful tool for metabolomics studies, and single cell MS (SCMS) analysis can provide unique information for understanding interactions among heterogeneous cells. In the current study, we utilized a direct co-culture system (with cell-cell contact) to study metabolomics of single cells affected by cell-cell interactions in their living status. A fluorescence microscope was utilized to distinguish these two types of cells for SCMS metabolomics studies using the Single-probe SCMS technique under ambient conditions. Our results show that through interactions with drug-resistant cells, drug-sensitive cancer cells acquired significantly increased drug resistance and exhibited drastically altered metabolites. Further investigation found that the increased drug resistance was associated with multiple metabolism regulations in drug-sensitive cells through co-culture such as the upregulation of sphingomyelins lipids and lactic acid and the downregulation of TCA cycle intermediates. The method allows for direct MS metabolomics studies of individual cells labeled with fluorescent proteins or dyes among heterogeneous populations.

Metabolomics studies of cell-cell interactions using single cell mass spectrometry combined with fluorescence microscopy.

Innate immunity has considerable specificity and can discriminate between individual species of microbes. In this regard, pathogens are "seen" as dangerous to the host and elicit an inflammatory response capable of destroying the microbes. This immune discrimination is achieved by toll-like receptors on host cells recognizing pathogens, such as , and microbe-specific pathogen-associated molecular pattern (PAMP) molecules, such as lipoteichoic acid (LTA). PAMPs impede wound healing by lengthening the inflammatory phase of healing and contributing to the development of chronic wounds. Preventing PAMPs from triggering the release of inflammatory cytokines will counteract the dysregulation of inflammation. Here, we use ELISA to evaluate the use of cationic molecules branched polyethylenimine (BPEI), PEGylated BPEI (PEG-BPEI), and polymyxin-B to neutralize anionic LTA and lower levels of TNF-α cytokine release from human THP-1 monocytes in a concentration-dependent manner. Additional data collected with qPCR shows that BPEI and PEG-BPEI reduce the expression profile of the TNF-α gene. Similar effects are observed for the neutralization of whole-cell  bacteria. In vitro cytotoxicity data demonstrate that PEGylation lowers the toxicity of PEG-BPEI (IC = 2661 μm) compared to BPEI (IC = 853 μM) and that both compounds are orders of magnitude less toxic than the cationic antibiotic polymyxin-B (IC = 79 μM). Additionally, the LTA neutralization ability of polymyxin-B is less effective than BPEI or PEG-BPEI. These properties of BPEI and PEG-BPEI expand their utility beyond disabling antibiotic resistance mechanisms and disrupting  biofilms, providing additional justification for developing these agents as wound healing therapeutics. The multiple mechanisms of action for BPEI and PEG-BPEI are superior to current wound treatment strategies that have a single modality.

Neutralizing  PAMPs that Trigger Cytokine Release from THP-1 Monocytes.

Firefighters are frequently exposed to a variety of chemicals formed from smoke, which pose a risk for numerous diseases, including cancer. Comparative urine proteome profiling could significantly improve our understanding of the early detection of potential cancer biomarkers. In this study, for the first time, we conducted a comparative protein profile analysis of 20 urine samples collected from ten real-life firefighters prior to and following emergency fire-induced smoke. Using a label-free quantitative proteomics platform, we identified and quantified 1325 unique protein groups, of which 45 proteins showed differential expressions in abundance in response to fire-smoke exposure (post) compared to the control (pre). Pathway analysis showed proteins associated with epithelium development (e.g., RHCG, HEG1, ADAMTSL2) and Alzheimer's disease (SORL1) were significantly increased in response to smoke exposure samples. A protein-protein-network study showed a possible link between these differentially abundant proteins and the known cancer gene (TP53). Moreover, a cross-comparison analysis revealed that seven proteins-ALDH1A1, APCS, POMC, COL2A1, RDX, DDAH2, and SDC4 overlapped with the previously published urine cancer proteome datasets, suggesting a potential cancer risk. Our findings demonstrated that the discovery proteomic platform is a promising analytical technique for identifying potential non-invasive biomarkers associated with fire-smoke exposure in firefighters that may be related to cancer.

Urine proteome profile of firefighters with exposure to emergency fire-induced smoke: A pilot study to identify potential carcinogenic effects.

The innate immune system is an evolutionarily conserved pathogen recognition mechanism that serves as the first line of defense against tissue damage or pathogen invasion. Unlike the adaptive immunity that recruits T-cells and specific antibodies against antigens, innate immune cells express pathogen recognition receptors (PRRs) that can detect various pathogen-associated molecular patterns (PAMPs) released by invading pathogens. Microbial molecular patterns, such as lipopolysaccharide (LPS) from Gram-negative bacteria, trigger signaling cascades in the host that result in the production of pro-inflammatory cytokines. LPS stimulation produces a strong immune response and excessive LPS signaling leads to dysregulation of the immune response. However, dysregulated inflammatory response during wound healing often results in chronic non-healing wounds that are difficult to control. In this work, we present data demonstrating partial neutralization of anionic LPS molecules using cationic branched polyethylenimine (BPEI). The anionic sites on the LPS molecules from Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are the lipid A moiety and BPEI binding create steric factors that hinder the binding of PRR signaling co-factors. This reduces the production of pro-inflammatory TNF-α cytokines. However, the anionic sites of Pseudomonas aeruginosa (P. aeruginosa) LPS are in the O-antigen region and subsequent BPEI binding slightly reduces TNF-α cytokine production. Fortunately, BPEI can reduce TNF-α cytokine expression in response to stimulation by intact P. aeruginosa bacterial cells and fungal zymosan PAMPs. Thus low-molecular weight (600 Da) BPEI may be able to counter dysregulated inflammation in chronic wounds and promote successful repair following tissue injury.

Low-Molecular Weight Branched Polyethylenimine Reduces Cytokine Secretion from Human Immune System Monocytes Stimulated with Bacterial and Fungal PAMPs.

Mung bean contains up to 32.6% protein and is one of the great sources of plant-based protein. Because many allergens also function as defense-related proteins, it is important to determine their abundance levels in the high-yielding, disease-resistant cultivars. In this study, for the first time, we compared the seed proteome of high-yielding mung bean cultivars developed by a conventional breeding approach. Using a label-free quantitative proteomic platform, we successfully identified and quantified a total of 1373 proteins. Comparative analysis between the high-yielding disease-resistant cultivar (MC5) and the other three cultivars showed that a total of 69 common proteins were significantly altered in their abundances across all cultivars. Bioinformatic analysis of these altered proteins demonstrated that PDF1 (a defensin-like protein) exhibited high sequence similarity and epitope matching with the established peanut allergens, indicating a potential mung bean allergen that showed a cultivar-specific response. Conversely, known mung bean allergen proteins such as PR-2/PR-10 (Vig r 1), Vig r 2, Vig r 4, LTP1, β-conglycinin, and glycinin G4 showed no alternation in the MC5 compared to other cultivars. Taken together, our findings suggest that the known allergen profiles may not be impacted by the conventional plant breeding method to develop improved mung bean cultivars.

Comparative Seed Proteome Profile Reveals No Alternation of Major Allergens in High-Yielding Mung Bean Cultivars.

Cell-cell interactions, which allow cells to communicate with each other through molecules in their microenvironment, are critical for the growth, health, and functions of cells. Previous studies show that drug-resistant cells can interact with drug-sensitive cells to elevate their drug resistance level, which is partially responsible for cancer recurrence. Studying protein targets and pathways involved in cell-cell communication provides essential information for fundamental cell biology studies and therapeutics of human diseases. In the current studies, we performed direct coculture and indirect coculture of drug-resistant and drug-sensitive cell lines, aiming to investigate intracellular proteins responsible for cell communication. Comparative studies were carried out using monoculture cells. Shotgun bottom-up proteomics results indicate that the P53 signaling pathway has a strong association with drug resistance mechanisms, and multiple TP53-related proteins were upregulated in both direct and indirect coculture systems. In addition, cell-cell communication pathways, including the phagosome and the HIF-signaling pathway, contribute to both direct and indirect coculture systems. Consequently, AK3 and H3-3A proteins were identified as potential targets for cell-cell interactions that are relevant to drug resistance mechanisms. We propose that the P53 signaling pathway, in which mitochondrial proteins play an important role, is responsible for inducing drug resistance through communication between drug-resistant and drug-sensitive cancer cells.

Proteomics Analysis of Interactions between Drug-Resistant and Drug-Sensitive Cancer Cells: Comparative Studies of Monoculture and Coculture Cell Systems.

Mass spectrometry (MS) has been one of the most widely used tools for bioanalytical analysis due to its high sensitivity, capability of quantitative analysis, and compatibility with biomolecules. Among various MS techniques, single cell mass spectrometry (SCMS) is an advanced approach to molecular analysis of cellular contents in individual cells. In tandem with the creation of novel experimental techniques, the development of new SCMS data analysis tools is equally important. As most published software packages are not specifically designed for pretreatment of SCMS data, including peak alignment and background removal, their applicability on processing SCMS data is generally limited. Hereby we introduce a Python platform, MassLite, specifically designed for rapid SCMS metabolomics data pretreatment. This platform is made user-friendly with graphical user interface (GUI) and exports data in the forms of each individual cell for further analysis. A core function of this tool is to use a novel peak alignment method that avoids the intrinsic drawbacks of traditional binning method, allowing for more effective handling of MS data obtained from high resolution mass spectrometers. Other functions, such as void scan filtering, dynamic grouping, and advanced background removal, are also implemented in this tool to improve pretreatment efficiency.

MassLite: An integrated python platform for single cell mass spectrometry metabolomics data pretreatment with graphical user interface and advanced peak alignment method.

Although most advanced-stage ovarian cancers initially respond to platinum- and taxane-based chemotherapy, the majority of them will recur and eventually develop chemoresistance. Among all drug resistance mechanisms, reduced drug uptake in tumors is regarded as an important pathway acquired by drug-resistant cancer cells. For patients with ovarian cancer, chemoresistant cells can develop into multicellular spheroids and spread through ascite fluid that accumulates in their abdomen. These spheroids consist of 3D structures that are highly heterogeneous with different shapes, sizes, and compositions of cell types. Thus, studying drug uptake at the single spheroid level is important for understanding chemosensitivity and chemoresistance; however, drug-uptake studies in single spheroids have not been previously reported due to the lack of a suitable analytical technique. In this study, we cultured spheroids using the ovarian cancer cell line (OVCAR-8) and treated them using paclitaxel or OSW-1, a natural compound with anticancer properties. We then developed a method of quantifying drug uptake in single spheroids using LC/MS measurements and then normalized the drug amount in each spheroid to its size and total protein content. Our method can be used in translational studies of drug development, treatment, and prediction of drug efficacy prior to chemotherapy.

Mass Spectrometry Quantification of Anticancer Drug Uptake in Single Multicellular Tumor Spheroids.

Chronic, low-grade inflammation is a hallmark of aging and various age-related diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). The prevalence of metabolic dysfunction-associated steatohepatitis (MASH), an advanced form of MASLD, increases with age and contributes to morbidity and mortality among the elderly. This study investigates the role of necroptosis, a programmed cell death pathway that promotes inflammation, in liver inflammaging and age-associated MASLD by utilizing genetic ablation models of two key necroptosis proteins, Mlkl or Ripk3. The absence of Mlkl or Ripk3 significantly reduced liver inflammation, steatosis, and fibrosis in aged male mice, supporting the role of necroptosis in age-associated MASLD. Additionally, Mlkl or Ripk3 deletion impacted other non-necroptotic cellular processes that drive inflammation and MASLD, such as cellular senescence, apoptosis, and autophagy in aged liver. Levels of plasma TNFα and IL6, key proinflammatory cytokines associated with inflammaging, are reduced in Mlkl or Ripk3 aged mice, supporting a systemic effect of necroptosis inhibition on inflammation. Proteomic analysis of liver tissues emphasizes the critical role of lipid and immune regulatory processes in maintaining liver homeostasis when Mlkl or Ripk3 is absent in aging liver. While Mlkl deletion did not affect the lifespan of mice, Ripk3 deletion shortened it. Additionally, Mlkl deficiency improved insulin sensitivity, whereas Ripk3 deficiency exacerbated glucose intolerance in aged mice. Thus, selective inhibition of Mlkl, not Ripk3, represents a potential therapeutic avenue for mitigating age-related liver disease and enhancing metabolic outcomes in the elderly.

Impact of Mlkl or Ripk3 deletion on age-associated liver inflammation, metabolic health, and lifespan.

The Single-probe single-cell mass spectrometry (SCMS) is an innovative analytical technique designed for metabolomic profiling, offering a miniaturized, multifunctional device capable of direct coupling to mass spectrometers. It is an ambient technique leveraging microscale sampling and nanoelectrospray ionization (nanoESI), enabling the analysis of cells in their native environments without the need for extensive sample preparation. Due to its miniaturized design and versatility, this device allows for applications in diverse research areas, including single-cell metabolomics, quantification of target molecules in single cell, MS imaging (MSI) of tissue sections, and investigation of extracellular molecules in live single spheroids. This review explores recent advancements in Single-probe-based techniques and their applications, emphasizing their potential utility in advancing MS methodologies in microscale bioanalysis.

Exploring Single-Probe Single-Cell Mass Spectrometry: Current Trends and Future Directions.

Single-cell mass spectrometry (SCMS) has emerged as a powerful tool for analyzing metabolites in individual cells, including live cells. However, cell metabolites have a rapid turnover rate, whereas maintaining metabolites' profiles of live cells during sample transport, storage, or extended measurements can be challenging. In this study, a cell preparation method, which integrates cell washing by volatile salt solution, rapid liquid nitrogen (LN) quenching, freeze-drying in vacuum, and freezer storage at -80 °C, to preserve cell metabolites for SCMS measurement is discussed. Experimental results revealed that LN quenching preserved the overall cell metabolome, whereas storage at -80 °C for 48 h slightly changed the metabolite profiles in quenched cells. However, metabolites in unquenched cells were changed regardless of low-temperature storage. The influence of omission of quenching and low-temperature storage on cell metabolites and relevant pathways were investigated. Results from this work indicate that cell quenching is necessary, but low-temperature storage time should be minimized to preserve cell metabolites. The method developed in the current work can be readily adopted by SCMS techniques with storage remaining largely unaltered, allowing for extended SCMS studies.

Developing a Cell Quenching Method to Facilitate Single Cell Mass Spectrometry Metabolomics Studies.

Seminal plasma (SP) primarily serves as the transport medium for sperm; however, protein and miRNA expression in SP have been found to be indicative of reproductive function and have been used to identify changes in spermatogenesis. It is also postulated that SP proteins and miRNAs play a role in direct communication with the female reproductive tract to alter uterine function and drive physiological changes that increase pregnancy success. Importantly, SP has been shown to be susceptible to paternal diet, indicating another method in which altered fetal programming may occur. Most work regarding the role of SP in pregnancy establishment as well as dietary contributions to SP composition has been conducted in mice and humans; however, the effects of diet on SP composition in livestock species warrants further investigation. Therefore, this study aims to investigate how paternal diet can alter the proteome and miRNA composition of SP from sheep to better understand their potential roles in male fertility and fetal programming. Here, we examined how a prepubertal methionine-enriched diet affected the miRNA and protein compositions of SP in Polypay rams. Comparative proteomics revealed that a total of 28 SP proteins were significantly increased, and 32 were decreased in abundance (P < 0.05, fold change > 1.5) in response to methionine supplementation. Additionally, 138 significant miRNAs were identified in the SP from treatment versus control rams, with 80 upregulated and 58 downregulated (P < 0.05, fold change > 2). Given that miRNAs are known regulators of mRNA expression, we performed a functional enrichment analysis of our differentially expressed miRNAs and found that 79 miRNAs target 37 proteins exhibiting differential abundance. Gene Ontology analysis revealed that targeted proteins were enriched in biological processes, including reproduction, fertilization, and embryo development. Overall, these results demonstrate that the SP composition of rams is susceptible to a methionine-enriched diet and may impact male fertility and offspring development.

Methionine supplementation-induced alteration of sheep seminal plasma miRNAs and proteome.

Colloidal all-inorganic lead halide perovskite quantum dots (QDs) are high-performance light-emitting materials with size-dependent optical properties and can be readily synthesized by mixing ionic precursors. However, the low formation energy of the perovskite lattice makes their growth too fast to control under regular reaction conditions. Diffusion-regulated CsPbBr perovskite QD growth is reported on a nanometer-sized liquid/liquid (L/L) interface supported in a micropipette tip without long-chain organic ligands. The precursors are divided into two immiscible solutions across the L/L interface to avoid additional nucleation, and the QD growth kinetics are regulated by the constrained cationic diffusion field depending on the size of the micropipette tip. QDs with unprecedentedly small sizes (2.7 nm) are obtained due to the slowed-down growth rates. The synthesis approach demonstrates the potential of micro-controlled colloidal QD synthesis for mechanistic studies and micro-fabrications.

ArticleTotal : 2	Year
Publication title Cited by 1	2025
Publication title	2026

Article	Year
Dimerization of 600 Da branched polyethylenimine improves β-lactam antibiotic potentiation against antibiotic-resistant Staphylococcus epidermidis and Pseudomonas aeruginosa. Chemical biology & drug design\| PubMed ID: 34923750	2023
Metabolomics studies of cell-cell interactions using single cell mass spectrometry combined with fluorescence microscopy. Chemical science\| PubMed ID: 35756524	2022
Neutralizing PAMPs that Trigger Cytokine Release from THP-1 Monocytes. ACS omega\| PubMed ID: 38463252	2024
Urine proteome profile of firefighters with exposure to emergency fire-induced smoke: A pilot study to identify potential carcinogenic effects. The Science of the total environment\| PubMed ID: 38583625	2024
Low-Molecular Weight Branched Polyethylenimine Reduces Cytokine Secretion from Human Immune System Monocytes Stimulated with Bacterial and Fungal PAMPs. ChemMedChem\| PubMed ID: 38740551	2024
Comparative Seed Proteome Profile Reveals No Alternation of Major Allergens in High-Yielding Mung Bean Cultivars. Journal of agricultural and food chemistry\| PubMed ID: 38836763	2024
Proteomics Analysis of Interactions between Drug-Resistant and Drug-Sensitive Cancer Cells: Comparative Studies of Monoculture and Coculture Cell Systems. Journal of proteome research\| PubMed ID: 38907724	2024
MassLite: An integrated python platform for single cell mass spectrometry metabolomics data pretreatment with graphical user interface and advanced peak alignment method. Analytica chimica acta\| PubMed ID: 39244309	2024
Mass Spectrometry Quantification of Anticancer Drug Uptake in Single Multicellular Tumor Spheroids. ACS pharmacology & translational science\| PubMed ID: 39416972	2024
Impact of Mlkl or Ripk3 deletion on age-associated liver inflammation, metabolic health, and lifespan. GeroScience\| PubMed ID: 39930289	2025
Exploring Single-Probe Single-Cell Mass Spectrometry: Current Trends and Future Directions. Analytical chemistry\| PubMed ID: 39999987	2025
Developing a Cell Quenching Method to Facilitate Single Cell Mass Spectrometry Metabolomics Studies. JACS Au\| PubMed ID: 40443889	2025
Methionine supplementation-induced alteration of sheep seminal plasma miRNAs and proteome. Journal of animal science\| PubMed ID: 40495297	2025
Regulating the Growth of Cesium Lead Bromide Quantum Dots at a Liquid/Liquid Interface Constrained in a Micropipette. Small methods\| PubMed ID: 40801159	2025

Zongkai Peng

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