Chronic type I interferon signaling promotes lipid-peroxidation-driven terminal CD8+ T cell exhaustion and curtails anti-PD-1 efficacy.

Identifying signals that govern the differentiation of tumor-infiltrating CD8+ T cells (CD8+ TILs) toward exhaustion can improve current therapeutic approaches for cancer. Here, we show that type I interferons (IFN-Is) act as environmental cues, enhancing terminal CD8+ T cell exhaustion in tumors. We find enrichment of IFN-I-stimulated genes (ISGs) within exhausted CD8+ T cells (Tex cells) in patients across various cancer types, with heightened ISG levels correlating with poor response to immune checkpoint blockade (ICB) therapy. In preclinical models, CD8+ TILs devoid of IFN-I signaling develop less exhaustion features, provide better tumor control, and show greater response to ICB-mediated rejuvenation. Mechanistically, chronic IFN-I stimulation perturbs lipid metabolism and redox balance in Tex cells, leading to aberrant lipid accumulation and elevated oxidative stress. Collectively, these defects promote lipid peroxidation, which potentiates metabolic and functional exhaustion of Tex cells. Thus, cell-intrinsic IFN-I signaling regulates the extent of CD8+ TIL exhaustion and has important implications for immunotherapy.

Differentiable Formation of Chiroptical Lanthanide Heterometallic Lnn Ln'4-n (L6 ) (n=0-4) Tetrahedra with C2 -Symmetrical Bis(tridentate) Ligands.

Construction of lanthanide heterometallic complex is important for engineering multifunction molecular containers. However, it remains a challenge because of the similar ionic radii of lanthanides. Herein we attempt to prepare chiral lanthanide heterometallic tetrahedra. Upon crystallization with a mixture of [Eu2 L3 ] and [Ln2 L3 ] (Ln=Gd, Tb and Dy) helicates, a mixture of heterometallic Eun Ln'4-n (L6 ) (n=0-4) tetrahedra was prepared. Selective formation of heterometallic tetrahedron was observed as MS deconvolution results deviated from statistical results. The formation of heterometallic tetrahedron was found to be sensitive to ionic radii as well as the ratio of the two helicates used in the crystallization.

Data storage using peptide sequences.

Humankind is generating digital data at an exponential rate. These data are typically stored using electronic, magnetic or optical devices, which require large physical spaces and cannot last for a very long time. Here we report the use of peptide sequences for data storage, which can be durable and of high storage density. With the selection of suitable constitutive amino acids, designs of address codes and error-correction schemes to protect the order and integrity of the stored data, optimization of the analytical protocol and development of a software to effectively recover peptide sequences from the tandem mass spectra, we demonstrated the feasibility of this method by successfully storing and retrieving a text file and the music file Silent Night with 40 and 511 18-mer peptides respectively. This method for the first time links data storage with the peptide synthesis industry and proteomics techniques, and is expected to stimulate the development of relevant fields.

Altered metabolites in guinea pigs with allergic asthma after acupoint sticking therapy: New insights from a metabolomics approach.

BACKGROUND: Clinical evidence gathered in Chinese communities suggested that acupoint sticking therapy could be an alternative treatment for asthma-related diseases. However, its underlying mechanism is still poorly understood. AIM/HYPOTHESIS: In this study, we aimed to investigate the mechanism of the anti-inflammatory effect of acupoint sticking application with 'Treatment of Winter Disease in Summer' (TWDS) prescription by using metabolomics. METHODS: Allergic asthma in guinea pig was sensitized and challenged by ovalbumin (OVA). Histopathological evaluation of the lung tissue was performed by hematoxylin and eosin (H&E) staining and Masson's trichrome staining. The levels of Th2 cytokine and IgE level in serum were measured using enzyme-linked immunoassay (ELISA). The mRNA expression levels of IL-4, IL-5, IL-13 and orosomucoid-like 3 (ORMDL3) were measured using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Proteins of NF-κB signaling pathway were measured using western blot. The serum metabolomics profiles were obtained by using ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS). RESULTS: The overall results confirmed that AST with TWDS prescription had a significant protective effect against OVA-induced allergic asthma in guinea pig. This treatment not only attenuated airway inflammation and collagen deposition in the airway, but also decreased the levels of IL-4, IL-5, IL-13 and IgE in serum. In addition, metabolomics results indicated that metabolisms of phospholipid, sphingolipid, purine, amino acid and level of epinephrine were restored back to the normal control level. Moreover, results of the gene expression of ORMDL3 in lung tissues indicated that AST using TWDS could alter the sphingolipid metabolism. Further western blotting analysis also showed that its anti-inflammatory mechanism was by decreasing the phosphorylation of p65 and IκB. CONCLUSION: The study demonstrated that metabolomics provides a better understanding of the actions of TWDS acupoint sticking therapy on OVA-induced allergic asthma.

The analysis of alpha-1-antitrypsin glycosylation with direct LC-MS/MS.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methodology has been developed to differentiate core- and antennary-fucosylated glycosylation of glycopeptides. Both the glycosylation sites (heterogeneity) and multiple possible glycan occupancy at each site (microheterogeneity) can be resolved via intact glycopeptide analysis. The serum glycoprotein alpha-1-antitrypsin (A1AT) which contains both core- and antennary-fucosylated glycosites was used in this study. Sialidase was used to remove the sialic acids in order to simplify the glycosylation microheterogeneity and to enhance the MS signal of glycopeptides with similar glycan structures. ß1-3,4 galactosidase was used to differentiate core- and antennary-fucosylation. In-source dissociation was found to severely affect the identification and quantification of glycopeptides with low abundance glycan modification. The settings of the mass spectrometer were therefore optimized to minimize the in-source dissociation. A three-step mass spectrometry fragmentation strategy was used for glycopeptide identification, facilitated by pGlyco software annotation and manual checking. The collision energy used for initial glycopeptide fragmentation was found to be crucial for improved detection of oxonium ions and better selection of Y1 ion (peptide+GlcNAc). Structural assignments revealed that all three glycosylation sites of A1AT glycopeptides contain complex N-glycan structures: site Asn70 contains biantennary glycans without fucosylation; site Asn107 contains bi-, tri- and tetra-antennary glycans with both core- and antennary-fucosylation; site Asn271 contains bi- and tri-antennary glycans with both core- and antennary-fucosylation. The relative intensity of core- and antennary-fucosylation on Asn107 was similar to that of the A1AT protein indicating that the glycosylation level of Asn107 is much larger than the other two sites.

Rapid differentiation of Ganoderma species by direct ionization mass spectrometry.

In this study, direct ionization mass spectrometry (DI-MS) has been developed for rapid differentiation of Ganoderma (known as Lingzhi in Chinese), a very popular and valuable herbal medicine. Characteristic mass spectra can be generated by DI-MS directly from the raw herbal medicines with the application of a high voltage and solvents. Rapid differentiation of the Ganoderma species that are officially stated in the Chinese pharmacopoeia from easily confused Ganoderma species could be achieved based on this method, as the acquired DI-MS spectra showed that ganoderic acids, the major active components of Ganoderma, could be found only in the official Ganoderma species but not in the confused Ganoderma species. In addition, classification of wild and cultivated Ganoderma and potential differentiation of Ganoderma from different geographical locations could be accomplished based on principal component analysis (PCA) or hierarchical clustering analysis (HCA). The method is rapid, simple and reproducible, and can be further extended to analysis of other herbal medicines.

Direct analysis of traditional Chinese medicines by mass spectrometry.

Analysis of traditional Chinese medicines (TCMs) plays important roles in quality control of TCMs and understanding their pharmacological effects. Mass spectrometry (MS) is a technique of choice for analysis of TCMs due to its superiority in speed, sensitivity and specificity. However, conventional MS analysis of TCMs typically requires extensive sample pretreatment and chromatographic separation, which could be time-consuming and laborious, prior to the analysis. The expanding usage of TCMs worldwide demands development of rapid, cost-effective and reliable methods for analysis of TCMs. In recent years, new sample preparation and ionization techniques have been developed to enable direct analysis of TCMs by MS, significantly reducing the analysis time and cost. In this review, various MS-based techniques, mainly including ambient ionization-MS and MALDI-MS based techniques, applied for direct analysis of TCMs are summarized and their applicability and future prospects are discussed.

Direct analysis of large living organism by megavolt electrostatic ionization mass spectrometry.

A new ambient ionization method allowing the direct chemical analysis of living human body by mass spectrometry (MS) was developed. This MS method, namely Megavolt Electrostatic Ionization Mass Spectrometry, is based on electrostatic charging of a living individual to megavolt (MV) potential, illicit drugs, and explosives on skin/glove, flammable solvent on cloth/tissue paper, and volatile food substances in breath were readily ionized and detected by a mass spectrometer.

Negative electrospray ionization on porous supporting tips for mass spectrometric analysis: electrostatic charging effect on detection sensitivity and its application to explosive detection.

The simplicity and easy manipulation of a porous substrate-based ESI-MS technique have been widely applied to the direct analysis of different types of samples in positive ion mode. However, the study and application of this technique in negative ion mode are sparse. A key challenge could be due to the ease of electrical discharge on supporting tips upon the application of negative voltage. The aim of this study is to investigate the effect of supporting materials, including polyester, polyethylene and wood, on the detection sensitivity of a porous substrate-based negative ESI-MS technique. By using nitrobenzene derivatives and nitrophenol derivatives as the target analytes, it was found that the hydrophobic materials (i.e., polyethylene and polyester) with a higher tendency to accumulate negative charge could enhance the detection sensitivity towards nitrobenzene derivatives via electron-capture ionization; whereas, compounds with electron affinities lower than the cut-off value (1.13 eV) were not detected. Nitrophenol derivatives with pKa smaller than 9.0 could be detected in the form of deprotonated ions; whereas polar materials (i.e., wood), which might undergo competitive deprotonation with the analytes, could suppress the detection sensitivity. With the investigation of the material effects on the detection sensitivity, the porous substrate-based negative ESI-MS method was developed and applied to the direct detection of two commonly encountered explosives in complex samples.

Electrospray ionization on porous spraying tips for direct sample analysis by mass spectrometry: enhanced detection sensitivity and selectivity using hydrophobic/hydrophilic materials as spraying tips.

RATIONALE: Despite various porous materials having been widely adopted as spraying tips for direct sample analysis using electrospray ionization mass spectrometry (ESI-MS), the effect of surface property and porosity of spraying tip materials on their analytical performances is not clear. Investigation of their relationships could provide insight into the proper choice and/or design of spraying tip materials for direct sample analysis. METHODS: The effect of spraying tip materials with different polarities, including polyester and polyethylene (hydrophobic) and wood (hydrophilic), on the detection sensitivity for a variety of compounds, and on the ESI onset voltage, were studied using ESI-MS. The porosity of each type of spraying tip was characterized by scanning electron microscopy (SEM). Factors governing the detection sensitivity were determined based on the correlation of the detection sensitivity to the ESI onset voltage, the polarity, and the porosity of the spraying tip materials. RESULTS: Hydrophobic tips (i.e., polyester and polyethylene) show better detection sensitivity for polar compounds but not for non-polar compounds, while hydrophilic tips (wooden tips) show the opposite effect. This phenomenon could be due to the difference in interaction between the analytes and the tips, causing the analytes to adsorb on the tip to different extents. In addition, the micro-porous nature of the tips could facilitate solvent diffusion for transporting analytes to the tip and maintain a stable spray for recording MS data. With the proper choice of spraying tip materials, trace amount of analytes at the picomole level can be detected with minimal sample pretreatment. CONCLUSIONS: Both the polarity and the porosity of the spraying tip materials could significantly affect detection sensitivity for a wide variety of analytes. With proper choice of spraying tip material, ESI on a porous spraying tip could be a sensitive method for the direct analysis of daily life samples.

Tissue-spray ionization mass spectrometry for raw herb analysis.

Tissue-spray ionization mass spectrometry is developed for the in situ chemical analysis of raw herbs under ambient conditions. We demonstrated that analyte molecules could be directly sprayed and ionized from solvent-wetted ginseng tissues upon the application of high electrical voltage to the tissue sample. Abundant phytochemicals/ metabolites, including ginsenosides, amino acids and oligosaccharides, could be detected from ginseng tissues when the tissue-spray experiments were conducted in positive ion mode. Thermally labile and easily hydrolyzed malonyl-ginsenosides were also detected in negative ion mode. The tissue-spray ionization method enables the direct detection of analytes from raw herb samples and preserves the sample integrity for subsequent morphological and/ or microscopic examination. In addition, this method is simple and fast for chemical profiling of wild-type and cultivated-type American ginsengs with differentiation.

Molecular imaging of banknote and questioned document using solvent-free gold nanoparticle-assisted laser desorption/ionization imaging mass spectrometry.

Direct chemical analysis and molecular imaging of questioned documents in a non/minimal-destructive manner is important in forensic science. Here, we demonstrate that solvent-free gold-nanoparticle-assisted laser desorption/ionization mass spectrometry is a sensitive and minimal destructive method for direct detection and imaging of ink and visible and/or fluorescent dyes printed on banknotes or written on questioned documents. Argon ion sputtering of a gold foil allows homogeneous coating of a thin layer of gold nanoparticles on banknotes and checks in a dry state without delocalizing spatial distributions of the analytes. Upon N(2) laser irradiation of the gold nanoparticle-coated banknotes or checks, abundant ions are desorbed and detected. Recording the spatial distributions of the ions can reveal the molecular images of visible and fluorescent ink printed on banknotes and determine the printing order of different ink which may be useful in differentiating real banknotes from fakes. The method can also be applied to identify forged parts in questioned documents, such as number/writing alteration on a check, by tracing different writing patterns that come from different pens.

Molecular histology analysis by matrix-assisted laser desorption/ionization imaging mass spectrometry using gold nanoparticles as matrix.

Gold nanoparticles (AuNPs) were applied and optimized as matrix for matrix-assisted laser desorption/ionization mass spectrometry analysis of animal tissues, and enabled histological analysis of animal tissues at molecular level by imaging mass spectrometry (IMS). AuNPs were coated on animal tissue in a solvent-free manner via argon ion sputtering. Metabolites, including neurotransmitters, fatty acids and nucleobases, were directly detected from mouse brain tissue. Based on region-specific chemical profiles, fine histological features of mouse brain tissue and heterogeneous regions of tumor tissue were both revealed.