µ-PESI-based MS profiling combined with untargeted metabolomics analysis for rapid identification of red wine geographical origin.

BACKGROUND: The commercial value of red wine is strongly linked to its geographical origin. Given the large global market, there is great demand for high-throughput screening methods to authenticate the geographical source of red wine. However, only limited techniques have been established up to now. RESULTS: Herein, a sensitive and robust method, namely probe electrospray ionization mass spectrometry (µ-PESI-MS), was established to achieve rapid analysis at approximately 1.2 min per sample without any pretreatment. A scotch near the needle tip provides a fixed micro-volume for each analysis to achieve satisfactory ion signal reproducibility (RSD < 26.7%). In combination with a machine learning algorithm, 16 characteristic ions were discovered from thousands of detected ions and were utilized for differentiating red wine origin. Among them, the relative abundances of two characteristic metabolites (trigonelline and proline) correlated with geographical conditions (sun exposure and water stress) were identified, providing the rationale for differentiation of the geographical origin. CONCLUSION: The proposed µ-PESI-MS-based method demonstrates a promising high-throughput determination capability in red wine traceability.

Covalent Bonding and Coulomb Repulsion-Guided AuNP Array: A Tunable and Reusable Substrate for Metabolomic Characterization of Lung Cancer Patient Sera.

Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) has gained increased attention in the metabolic characterization of human biofluids. However, the stability and reproducibility of nanoparticle-based substrates remain two of the biggest challenges in high-salt environments. Here, by controlling the extent of Coulomb repulsion of 26 nm positively charged AuNPs, a homogeneous layer of covalently bonded AuNPs on a coverslip with tunable interparticle distances down to 16 nm has been successfully fabricated to analyze small biomolecules in human serum. Compared with the self-assembled AuNP array, the covalently bonded AuNP array showed superior performances on stability, reproducibility, and sensitivity in high-salt environments. The stable attachment of AuNPs maintained a detection reproducibility with a RSD less than 12% and enabled the reusability of the array for 10 experiments without significant signal deterioration (<15%) and carryover effects. Moreover, the closely positioned AuNPs allowed the coupling of photoinduced plasmons to generate an enhanced electric field, which promotes the generation of excited electrons to facilitate the desorption/ionization processes instead of the heat dissipation, thus enhancing the detection sensitivity with detection limits down to the femtomole level. Combined with machine learning methods, the AuNP array has been successfully applied to discover seven biomarkers for differentiating early-stage lung cancer patients from healthy controls. It is anticipated that this simple approach of developing robust AuNP arrays can also be extended to other types of NP arrays for wider applications of SALDI-MS technology.

Sensitive photoelectrochemical detection of colitoxin DNA based on NCDs@CuO/ZnO heterostructured nanocomposites with efficient separation capacity of photo-induced carriers.

A metal-organic framework (MOF) of Cu-TPA (terephthalic acid) microsphere was prepared, followed by calcinating the MOF precursor of Cu-TPA/ZIF-8 mixture to obtain the CuO/ZnO. N-doped carbon dots (NCDs) were employed to combine the CuO/ZnO composite to form a tripartite heterostructured architecture of NCDs@CuO/ZnO, which led to a fierce enlargement of the photocurrent response. This  was ascribed to the thinner-shell structure of the CuO microsphere and the fact that hollow ZnO particles could sharply promote the incidence intensity of visible light. The more porous defectiveness exposed on CuO/ZnO surface was in favor of rapidly infiltrating electrolyte ions. The p-n type CuO/ZnO composite with more contact interface could abridge the transfer distance of photo-induced electron (e-1)/hole (h+) pairs and repress their recombination availably. NCDs not only could boost electron transfer rate on the electrode interface but also successfully sensitized the CuO/ZnO composite, which resulted in high conversion efficiency of photon-to-electron. The probe DNA (S1) was firmly assembled on the modified ITO electrode surface (S1/NCDs@CuO/ZnO) through an amidation reaction. Under optimal conditions, the prepared DNA biosensor displayed a wide linear range of 1.0 × 10-6 ~ 7.5 × 10-1 nM and a low limit of detection (LOD) of 1.81 × 10-7 nM for colitoxin DNA (S2) measure, which exhibited a better photoelectrochemistry (PEC) analysis performance than that obtained by differential pulse voltammetry techniques. The relative standard deviation (RSD) of the sensing platform for target DNA detection of 5.0 × 10-2 nM was 6.3%. This proposed DNA biosensor also showed good selectivity, stability, and reproducibility, demonstrating that the well-designed and synthesized photoactive materials of NCDs@CuO/ZnO are promising candidates for PEC analysis.

Sensitive Detection of Separated Charges in Nanohybrids by Laser Excitation Mass Spectrometry with Tetrabutylammonium Cationic Probe.

Charge separation lays the foundation for photocatalysis and photovoltaics, in which the catalytic/voltaic efficiency is primarily related to the amount of separated charges generated. Yet, direct experimental approaches for the quantification of separated charges are very limited, especially for nanostructures in small quantities. Here, by laser excitation mass spectrometry with tetrabutylammonium as a sensitive probe, the separated charges in gold-metal sulfide core-shell nanostructures are determined and correlated with the bandgap of the semiconductor shell. Moreover, the separated charges formed can already be detected unambiguously in only an attomole-level of nanoparticles (i.e., 1 × 108 NPs).

The Hidden Heroes: Holes in Charge-Driven Desorption Mass Spectrometry.

Photoexcited hot carriers have high impacts on various fields. However, in contrast to the attention attracted by the hot-electrons, the contributions of holes are seldom recognized. Yet, by simply removing the hot-electrons but retaining the holes on the surface of photoexcited plasmonic gold nanoparticles, a 60-fold ion intensity amplification could be achieved in laser desorption ionization mass spectrometry. A new desorption mechanism termed "charge-driven desorption" has been derived to rationalize the significant improvement.

Surface optimization of gold nanoparticle mass tags for the sensitive detection of protein biomarkers via immuno-capture LI-MS.

HPV-induced cervical cancer is one of the most lethal cancers. Therefore, the development of a reliable and accurate method for the early diagnosis of HPV infections is highly important. Here, gold nanoparticles (AuNPs) were utilized as mass tags in an immuno-capture LI-MS assay for the detection of HPV marker proteins. Through the optimization of the amount of antibodies and surface charges on AuNPs, high antigen detection efficiency with minimal non-specific binding was achieved. With optimized antibody-conjugated AuNPs, low attomole amount of HPV proteins in HeLa cell lysate was quantified.

Rapid labeling of intracellular His-tagged proteins in living cells.

Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni(2+)-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni(2+)-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni(2+) ions. The probe, driven by Ni(2+)-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

Silver-gold alloy nanoparticles as tunable substrates for systematic control of ion-desorption efficiency and heat transfer in surface-assisted laser desorption/ionization.

Systematically controlling heat transfer in the surface-assisted laser desorption/ionization (SALDI) process and thus enhancing the analytical performance of SALDI-MS remains a challenging task. In the current study, by tuning the metal contents of Ag-Au alloy nanoparticle substrates (AgNPs, Ag55Au45NPs, Ag15Au85NPs and AuNPs, ∅: ∼2.0 nm), it was found that both SALDI ion-desorption efficiency and heat transfer can be controlled in a wide range of laser fluence (21.3 mJ cm-2 to 125.9 mJ cm-2). It was discovered that ion detection sensitivity can be enhanced at any laser fluence by tuning up the Ag content of the alloy nanoparticle, whereas the extent of ion fragmentation can be reduced by tuning up the Au content. The enhancement effect of Ag content on ion desorption was found to be attributable to the increase in laser absorption efficiency (at 355 nm) with Ag content. Tuning the laser absorption efficiency by changing the metal composition was also effective in controlling the heat transfer from the NPs to the analytes. The laser-induced heating of Ag-rich alloy NPs could be balanced or even overridden by increasing the Au content of NPs, resulting in the reduction of the fragmentation of analytes. In the correlation of experimental measurement with molecular dynamics simulation, the effect of metal composition on the dynamics of the ion desorption process was also elucidated. Upon increasing the Ag content, it was also found that phase transition temperatures, such as melting, vaporization and phase explosion temperature, of NPs could be reduced. This further enhanced the desorption of analyte ions via phase-transition-driven desorption processes. The significant cooling effect on the analyte ions observed at high laser fluence was also determined to be originated from the phase explosion of the NPs. This study revealed that the development of alloy nanoparticles as SALDI substrates can constitute an effective means for the systematic control of ion-desorption efficiency and the extent of heat transfer, which could potentially enhance the analytical performance of SALDI-MS.

A novel mechanism of XIAP degradation induced by timosaponin AIII in hepatocellular carcinoma.

Inducing tumor cell death is one of the major therapeutic strategies in treating cancer. The aim of this study is to investigate the mechanism underlying the involvement of autophagy in cell death induced by timosaponin AIII (TAIII). Cell viability was determined by MTT and cologenic assay; apoptosis was determined by flow cytometry and TUNEL assay; autophagy was examined by immunoblotting and immunofluorescence; ubiquitination was detected by co-immunoprecipitation; mRNA expression was detected by real-time PCR; and determination of necrotic cell death was approached with LDH assay. The in vivo tumor growth inhibition was determined by xenograft model. TAIII exhibits potent cytotoxicity on human hepatocellular carcinoma (HCC) cells without severe hepatic toxicity. TAIII induced caspase-dependent apoptosis in HCC, and the induction of apoptosis was attributed to the inhibition of TAIII on XIAP expression. Repressing XIAP expression allowed cell tolerance toward the treatment with TAIII. The suppression of XIAP by TAIII is under post-transcriptional control and independent of proteasomal-driven proteolysis. Instead, TAIII-induced AMPKα/mTOR-dependent autophagy was responsible for XIAP suppression and triggered the XIAP heading lysosomal degradation pathway. Ubiquitination of IAPs is required for the autophagic degradation induced by TAIII. Blockade of autophagy turns on the switch of necrotic cell death in TAIII-treated cells. Timosaponin AIII induces HCC cell apoptosis through a p53-independent mechanism involving XIAP degradation through autophagy-lysosomal pathway. The possibility of developing TAIII as a new anti-tumor agent is worth considering.

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.

Hierarchical superstructure aerogels for in situ biofluid metabolomics.

High-throughput biofluid metabolomics analysis for screening life-threatening diseases is urgently needed. However, the high salt content of biofluid samples, which introduces severe interference, can greatly limit the analysis throughput. Here, a new 3-D interconnected hierarchical superstructure, namely a "plasmonic gold-on-silica (Au/SiO2) double-layered aerogel", integrating distinctive features of an upper plasmonic gold aerogel with a lower inert silica aerogel was successfully developed to achieve in situ separation and storage of inorganic salts in the silica aerogel, parallel enrichment of metabolites on the surface of the functionalized gold aerogel, and direct desorption/ionization of enriched metabolites by the photo-excited gold aerogel for rapid, sensitive, and comprehensive metabolomics analysis of human serum/urine samples. By integrating all these unique advantages into the hierarchical aerogel, multifunctional properties were introduced in the SALDI substrate to enable its effective utilization in clinical metabolomics for the discovery of reliable metabolic biomarkers to achieve unambiguous differentiation of early and advanced-stage lung cancer patients from healthy individuals. This study provides insight into the design and application of superstructured nanomaterials for in situ separation, storage, and photoexcitation of multi-components in complex biofluid samples for sensitive analysis.

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.

Integration of Non-targeted Proteomics Mass Spectrometry with Machine Learning for Screening Cooked Beef Adulterated Samples.

The degradation of ingredients in heat-processed meat products makes their authentication challenging. In this study, protein profiles of raw beef, chicken, duck, pork, and binary simulated adulterated beef samples (chicken-beef, duck-beef, and pork-beef) and their heat-processed samples were obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Heat-stable characteristic proteins were found by screening the overlapping characteristic protein ion peaks of the raw and corresponding heat-processed samples, which were discovered by partial least-squares discriminant analysis. Based on the 36 heat-stable characteristic proteins, qualitative classification for the raw and heat-processed meats was achieved by extreme gradient boosting. Moreover, quantitative analysis via partial least squares regression was applied to determine the adulteration ratio of the simulated adulterated beef samples. The validity of the approach was confirmed by a blind test with the mean accuracy of 97.4%. The limit of detection and limit of quantification of this method were determined to be 5 and 8%, respectively, showing its practical aspect for the beef authentication.

Serum Lipidomic Fingerprints Encode Early Diagnosis and Staging of Lung Cancer on a Novel PbS/Au-Layered Substrate.

Lung cancer (LC) is a major cause of mortality among malignant tumors. Early diagnosis through lipidomic profiling can improve prognostic outcomes. In this study, a uniform PbS/Au-layered substrate that enhances the laser desorption/ionization process, an interfacial process triggered on the substrate surface upon laser excitation, was designed to efficiently characterize the lipidomic profiles of LC patient serum. By controlling the stacking arrangement and particle sizes of PbS QDs and AuNPs, the optimized substrate promotes the generation of excited electrons and creates an enhanced electric field that polarizes analyte molecules, facilitating ion adduction formation ([M + Na]+ and [M + K]+) and enhancing detection sensitivity down to the femtomole level. Combining multivariate statistics and machine learning, a distinct lipidomic biomarker panel is successfully identified for the early diagnosis and staging of LC, with an accurate prediction validated by an area under the curve of 0.9479 and 0.9034, respectively. We also found that 18 biomarkers were significantly correlated with six metabolic pathways associated with LC. These results demonstrate the potential of this innovative PbS/Au-layered substrate as a sensitive platform for accurate diagnosis of LC and facilitate the development of lipidomic-based diagnostic tools for other cancers.

A high-symmetry coordination cage from 38- or 62-component self-assembly.

Artificial molecular architecture from a large number of subcomponents (>50) via self-assembly remains a formidable challenge for chemists. Reaction of 38 components [14 Ni(2+) and 24 N-methyl-1-(4-imidazolyl)methanimine] under solvothermal conditions reproducibly leads to the formation of a high-symmetry coordination cage. This polyhedral cage can also be obtained in high yield by self-assembly of 62 commercially available subcomponents (24 methylamine, 24 4-formylimidazole, and 14 Ni(2+)) under mild conditions involving synchronized formation of both dynamic covalent bonds and coordination bonds. Guest molecules (e.g., water, methylamine, and methanol) are randomly imprisoned in the cage.

Combining MALDI-MS with machine learning for metabolomic characterization of lung cancer patient sera.

An increasing amount of evidence has proven that serum metabolites can instantly reflect disease states. Therefore, sensitive and reproducible detection of serum metabolites in a high-throughput manner is urgently needed for clinical diagnosis. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a high-throughput platform for metabolite detection, but it is hindered by significant signal fluctuations because of the "sweet spot" effect of organic matrices. Here, by screening two transformation methods and four normalization techniques to reduce the significant signal fluctuations of the DHB matrix, an integrated MALDI-MS data processing approach combined with machine learning methods was established to reveal metabolic biomarkers of lung cancer. In our study, 13 distinctive features with statistically significant differences (p < 0.001) between 34 lung cancer patients and 26 healthy controls were selected as significant potential biomarkers of lung cancer. 6 out of the 13 distinctive features were identified as intact metabolites. Our results demonstrate the potential for clinical application of MALDI-MS in serum metabolomics for biomarker screening in lung cancer.

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.

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 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.

Harvesting More Energetic Photoexcited Electrons from Closely Packed Gold Nanoparticles.

The characterization of photoexcited electrons on the surface of nanomaterial remains challenging. Herein, laser excitation mass spectrometry combined with a chemical thermometer and electron acceptor has been developed to characterize the energetics and population density of photoexcited electrons transferred from gold nanoparticles (AuNPs). In contrast to laser fluence and bias voltage, the hot spots of closely packed AuNPs play a more significant role in enhancing the average energetics of photoexcited electrons, which can be harvested effectively by the electron acceptor. By harvesting more energetic photoexcited electrons for the desorption and ionization process, it is anticipated that the sensitive detection of biomarkers can be achieved, which is beneficial to metabolomic studies and early disease diagnosis.