Pocket-Size Wireless Nanoelectrospray Ionization Mass Spectrometry for Metabolic Analysis of Salty Biofluids and Single Cells.

Analysis of volume-limited biological samples such as single cells and biofluids not only benefits clinical purposes but also promotes fundamental research in life sciences. Detection of these samples, however, imposes strict requirements on measurement performance because of the minimal volume and concentrated salts of the samples. Herein, we developed a self-cleaning nanoelectrospray ionization device powered by a pocket-size "MasSpec Pointer" (MSP-nanoESI) for metabolic analysis of salty biological samples with limited volume. The self-cleaning effect induced by Maxwell-Wagner electric stress helps with keeping the borosilicate glass capillary tip free from clogging and thus increasing salt tolerance. This device possesses a high sample economy (about 0.1 µL per test) due to its pulsed high voltage supply, sampling method (dipping the nanoESI tip into analyte solution), and contact-free electrospray ionization (ESI) (the electrode does not touch the analyte solution during ESI). High repeatable results could be acquired by the device with a relative standard deviation (RSD) of 1.02% for voltage output and 12.94% for MS signals of caffeine standard. Single MCF-7 cells were metabolically analyzed directly from phosphate buffered saline, and two types of untreated cerebrospinal fluid from hydrocephalus patients were distinguished with 84% accuracy. MSP-nanoESI gets rid of the bulky apparatus and could be held in hand or put into one's pocket for transportation, and it could operate for more than 4 h without recharge. We believe this device will boost scientific research and clinical usage of volume-limited biological samples with high-concentration salts in a low-cost, convenient, and rapid manner.

Revealing the In Situ Behavior of Aggregation-Induced Emission Nanoparticles and Their Biometabolic Effects via Mass Spectrometry Imaging.

Simultaneous imaging of exogenous nanomaterials and endogenous metabolites in situ remains challenging and is beneficial for a systemic understanding of the biological behavior of nanomaterials at the molecular level. Here, combined with label-free mass spectrometry imaging, visualization and quantification of the aggregation-induced emission nanoparticles (NPs) in tissue were realized as well as related endogenous spatial metabolic changes simultaneously. Our approach enables us to identify the heterogeneous deposition and clearance behavior of nanoparticles in organs. The accumulation of nanoparticles in normal tissues results in distinct endogenous metabolic changes such as oxidative stress as indicated by glutathione depletion. The low passive delivery efficiency of nanoparticles to tumor foci suggested that the enrichment of NPs in tumors did not benefit from the abundant tumor vessels. Moreover, spatial-selective metabolic changes upon NPs mediated photodynamic therapy was identified, which enables understanding of the NPs induced apoptosis in the process of cancer therapy. This strategy allows us to simultaneously detect exogenous nanomaterials and endogenous metabolites in situ, hence to decipher spatial selective metabolic changes in drug delivery and cancer therapy processes.

Biofluids Metabolic Profiling Based on PS@Fe3O4-NH2 Magnetic Beads-Assisted LDI-MS for Liver Cancer Screening.

Liver cancer (LC) is the third frequent cause of death worldwide, so early diagnosis of liver cancer patients is crucial for disease management. Herein, we applied NH2-coated polystyrene@Fe3O4 magnetic beads (PS@Fe3O4-NH2 MBs) as a matrix material in laser desorption/ionization mass spectrometry (LDI-MS). Rapid, sensitive, and selective metabolic profiling of the native biofluids was achieved without any inconvenient enrichment or purification. Then, based on the selected m/z features, LC patients were discriminated from healthy controls (HCs) by machine learning, with the high area under the curve (AUC) values for urine and serum assessments (0.962 and 0.935). Moreover, initial-diagnosed and subsequent-visited LC patients were also differentiated, which indicates potential applications of this method in early diagnosis. Furthermore, among these identified compounds by FT-ICR MS, the expression level of some metabolites changed from HCs to LCs, including 29 and 12 characteristic metabolites in human urine and serum samples, respectively. These results suggest that PS@Fe3O4-NH2 MBs-assisted LDI-MS coupled with machine learning is feasible for LC clinical diagnosis.

Profiling of Urine Carbonyl Metabolic Fingerprints in Bladder Cancer Based on Ambient Ionization Mass Spectrometry.

The diagnosis of bladder cancer (BC) is currently based on cystoscopy, which is invasive and expensive. Here, we describe a noninvasive profiling method for carbonyl metabolic fingerprints in BC, which is based on a desorption, separation, and ionization mass spectrometry (DSI-MS) platform with N,N-dimethylethylenediamine (DMED) as a differential labeling reagent. The DSI-MS platform avoids the interferences from intra- and/or intersamples. Additionally, the DMED derivatization increases detection sensitivity and distinguishes carboxyl, aldehyde, and ketone groups in untreated urine samples. Carbonyl metabolic fingerprints of urine from 41 BC patients and 41 controls were portrayed and 9 potential biomarkers were identified. The mechanisms of the regulations of these biomarkers have been tentatively discussed. A logistic regression (LR) machine learning algorithm was applied to discriminate BC from controls, and an accuracy of 85% was achieved. We believe that the method proposed here may pave the way toward the point-of-care diagnosis of BC in a patient-friendly manner.

Mass Spectrometry Imaging Reveals In Situ Behaviors of Multiple Components in Aerosol Particles.

The inhalation of atmospheric particles is deleterious to human health. However, as a complex mixture, tracing the behaviors of multiple components from real aerosol particles is crucial but unachievable by the existing methods. Here, taking advantage of the intrinsic fingerprints of elemental carbon (EC) and organic carbon (OC) in carbonaceous aerosol (CA) upon laser irradiation, we proposed a label-free mass spectrometry imaging method to visualize and quantify the deposition, translocation and component variation of CA in organs. With this method, the heterogeneous deposition, clearance and release behavior of CA in lung, that more OC was released in parenchyma and OC was cleared faster than EC, was observed. The translocation of CA to extrapulmonary organs including kidney, liver, spleen and even brain was also verified and quantified. By comparing the ratio of OC to EC, an organ-specific release behavior of OC from CA during circulation was revealed. In orthotopic lung and liver tumor, OC was found to penetrate more into tumor foci than EC. This technique provides deeper information for understanding the systemic health effects of aerosol particles.

Application of Graphdiyne in Surface-Assisted Laser Desorption Ionization Mass Spectrometry.

Graphdiyne (GD) is a new kind of carbon nanomaterial which has carbon-carbon triple bonds to form a layered structure. Here, we report the application of GD as the matrix for small molecule analysis in laser desorption ionization mass spectrometry (LDI MS). The GD matrix displayed two advantages: little background in the low mass range and good molecular ion signal in negative ion mode for many small molecules, e.g., fatty acids, amino acids, peptides, and drugs can be obtained in negative ion mode. By comparing the signal intensity of tetraphenylborate and juglone with and without GD existing, it was found that GD can enhance both of the desorption efficiency and ionization efficiency in LDI. Through analysis of the serum samples from liver cancer patients and healthy people, the GD-assisted LDI MS results showed that fatty acids could be used as potential biomarkers for the early diagnosis of liver cancer.

Mass, Size, and Density Measurements of Microparticles in a Quadrupole Ion Trap.

The physical properties of microparticles, such as mass, size, and density, are critical for their functions. The comprehensive characterization of these physical parameters, however, remains a fundamental challenge. Here, we developed a particle mass spectrometry (PMS) methodology for determining the mass, size, and density of microparticles simultaneously. The collisional cross-section (CCS) and mass spectrometry (MS) measurements were performed in a single quadrupole ion trap (QIT), and the two modes can be switched easily by tuning the electric and gas hydrodynamic fields of the QIT. The feasibility of the method was demonstrated through a series of monodispersed polystyrene (PS) and silica (SiO2) particle standards. The SiO2/polypyrrole core-shell particles were also successfully characterized, and the measured results were verified by using conventional methods.

Laser cleavable probes for in situ multiplexed glycan detection by single cell mass spectrometry.

Glycans binding on the cell surface through glycosylation play a key role in controlling various cellular processes, and glycan analysis at a single-cell level is necessary to study cellular heterogeneity and diagnose diseases in the early stage. Herein, we synthesized a series of laser cleavable probes, which could sensitively detect glycans on single cells and tissues by laser desorption ionization mass spectrometry (LDI-MS). This multiplexed and quantitative glycan detection was applied to evaluate the alterations of four types of glycans on breast cancer cells and drug-resistant cancer cells at a single-cell level, indicating that drug resistance may be related to the upregulation of glycan with a ß-d-galactoside (Galß) group and Neu5Aca2-6Gal(NAc)-R. Moreover, the glycan spatial distribution in cancerous and paracancerous human tissues was also demonstrated by MS imaging, showing that glycans are overexpressed in cancerous tissues. Therefore, this single-cell MS approach exhibits promise for application in studying glycan functions which are essential for clinical biomarker discovery and diagnosis of related diseases.

Mass spectrometry imaging of the in situ drug release from nanocarriers.

It is crucial but of a great challenge to study in vivo and in situ drug release of nanocarriers when developing a nanomaterial-based drug delivery platform. We developed a new label-free laser desorption/ionization mass spectrometry (MS) imaging strategy that enabled visualization and quantification of the in situ drug release in tissues by monitoring intrinsic MS signal intensity ratio of loaded drug over the nanocarriers. The proof of concept was demonstrated by investigating the doxorubicin (DOX)/polyethylene glycol-MoS2 nanosheets drug delivery system in tumor mouse models. The results revealed a tissue-dependent release behavior of DOX during circulation with the highest dissociation in tumor and lowest dissociation in liver tissues. The drug-loaded MoS2 nanocarriers are predominantly distributed in lung, spleen, and liver tissues, whereas the accumulation in the tumor was unexpectedly lower than in normal tissues. This new strategy could also be extended to other drug-carrier systems, such as carbon nanotubes and black phosphorus nanosheets, and opened a new path to evaluate the drug release of nanocarriers in the suborgan level.

A novel mass spectrometry method based on competitive non-covalent interaction for the detection of biomarkers.

We report a novel biosensor platform based on competitive non-covalent interaction between ssDNA and a mass tag towards AuNPs, which detects PSA biomarkers sensitively, observed using MALDI MS. A detection limit of 57 pg mL-1 has been achieved, showing an improvement of two orders of magnitude compared to the traditional spectroscopic method.

Laser Cleavable Probes-Based Cell Surface Engineering for in Situ Sialoglycoconjugates Profiling by Laser Desorption/Ionization Mass Spectrometry.

Cell-surface sialoglycoconjugates (sialoglycoproteins and sialoglycolipids) play important roles in cell-cell interactions and related tumor metastasis process. Although there have been some analytical methods to evaluate the sialoglycoconjugates, an effective method providing both qualitative and quantitative information is still deficient. Here we establish an extraction-free, sensitive, and high-throughput platform to realize in situ detection of the cell-surface sialoglycoconjugates on various cell lines, e.g., cancer and normal cells by laser desorption/ionization mass spectrometry (LDI MS). In this proposal, azide groups were introduced into the ends of cell-surface sialoglycoconjugates by the biorthogonal method, and then the sialoglycoconjugates were armed with a laser-cleavable probe (Tphsene) through click chemistry. We can easily get the probes signal under laser irradiation, which reflected the presence of cell-surface sialoglycoconjugates. Different cell lines were discriminated simultaneously, and the LDI relative quantification agreed with fluorescent results. Besides, a linear quantitation relationship in the range of 100 fmol to 100 pmol was obtained with a designed and synthesized internal standard (phTsane) added. A detection limit of 5 fmol was obtained with good reproducibility. Based on the quantitative and high-throughput ability, we conducted pharmacodynamics study of drug (tunicamycin) on cancer cells. In addition, we found the tag was safe from sweet-spot effect of matrix adding. The simultaneous detection of sialoglycoconjugates and metabolites was therefore achieved. We believe that this laser cleavable probes-based cell-surface engineering for sialoglycoconjugates platform means great significance to diagnosis, prognosis, and therapeutic purposes. Besides, this strategy can be applied to other glycoconjugates which is hard to detect and the related disease processes when more corresponding chemically modified sugar substrates and exact biorthogonal reactions are developed.

N-Phenyl-2-naphthylamine as a Novel MALDI Matrix for Analysis and in Situ Imaging of Small Molecules.

Due to its strong ultraviolet absorption, low background interference in the small molecular range, and salt tolerance capacity, N-phenyl-2-naphthylamine (PNA) was developed as a novel matrix in the present study for analysis and imaging of small molecules by matrix-assisted laser desorption/ionization mass spectrometry time-of-fight (MALDI-TOF MS). The newly developed matrix displayed good performance in analysis of a wide range of small-molecule metabolites including free fatty acids, amino acids, peptides, antioxidants, and phospholipids. In addition, PNA-assisted LDI MS imaging of small molecules in brain tissue of rats subjected to middle cerebral artery occlusion (MCAO) revealed unique distributions and changes of 89 small-molecule metabolites including amino acids, antioxidants, free fatty acids, phospholipids, and sphingolipids in brain tissue 24 h postsurgery. Fifty-nine of the altered metabolites were identified, and all the changed metabolites were subject to relative quantitation and statistical analysis. The newly developed matrix has great potential application in the field of biomedical research.

Development of Visible-Wavelength MALDI Cell Mass Spectrometry for High-Efficiency Single-Cell Analysis.

Mass is a fundamental physical property of an individual cell, from which is revealed the cell growth, cycle, and activity. Taking advantage of cell mass spectrometry (CMS), accurate mass measurement of a charged single cell has been achieved. However, with the increasing demand for high-efficiency single-cell analysis in biology, the limited throughput and inefficient cell desorption/ionization of the CMS inevitably become important issues. To address the challenge, a state of the art visible-wavelength matrix assisted laser desorption/ionization (MALDI) CMS was developed. The employed transmission mode laser ablation and fast evaporation sample preparation enabled the visible-wavelength MALDI to be soft enough and to generate intact charged cells for mass measurement. By using resorufin as matrix, ten sorts of cells, viz., red blood cells (RBCs), Jurkat (JK), CCRF-CEM, SNU-5, BGC-803, MCF-7, L-O2, 293T, Hep G2, and A549 cells, have been successfully analyzed. It was found that the desorption/ionization efficiency of visible-wavelength MALDI was at least 3-fold higher than that of conventional laser-induced acoustic desorption (LIAD) and relevant to the suspension/adherent property of analyzed cells. Based on the measured mass, different cell types in either the individual or mixed state can be differentiated successfully.

In situ bioconjugation and ambient surface modification using reactive charged droplets.

Molecular ions are generated in induced electrospray ionization, and they can be transported to grounded ambient surfaces in the form of charged microdroplets. Efficient amide bonds formation between amines and carboxylic acids were observed inside charged droplets during transfer to the surface. Biomolecules derivatized using this method were self-assembled on a bare gold surface via Au-S bonds under the charged microdroplet environment. Cyclic voltammetric analysis of the self-assembled molecular film showed accelerated protein derivatization with cysteine, which allowed the covalent immobilization of the protein to the gold surface. Cytochrome C-functionalized electrodes prepared using the induced dual nanoelectrospray process showed bioactivity toward aqueous solutions of hydrogen peroxide below 50 µM. In effect, we have developed a method that allows derivatization of biomolecules and their immobilization at ambient surfaces in a single experimental step.

MALDI-TOF MS imaging of metabolites with a N-(1-naphthyl) ethylenediamine dihydrochloride matrix and its application to colorectal cancer liver metastasis.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is a label-free technique for identifying multiplex metabolites and determining both their distribution and relative abundance in situ. Our previous study showed that N-(1-naphthyl) ethylenediamine dihydrochloride (NEDC) could act as a matrix for laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) detection of oligosaccharides in solution. In the present study, NEDC-assisted LDI-TOF MSI yielded many more endogenous compound peaks between m/z 60 and m/z 1600 than 9-aminoacridine (9-AA). Our results show that NEDC-assisted LDI-TOF MSI is especially well-suited for examining distributions of glycerophospholipids (GPs) in addition to low molecular weight metabolites below m/z 400. Particularly, NEDC matrix allowed the LDI-TOF MSI of glucose in animal tissue. Furthermore, NEDC-assisted LDI-TOF MSI was applied to a mouse model of colorectal cancer liver metastasis. We revealed the distinct spatio-molecular signatures of many detected compounds in tumor or tumor-bearing liver, and we found that taurine, glucose, and some GPs decreased in tumor-bearing liver as the tumor developed in liver. Importantly, we also found a glucose gradient in metastatic tumor foci for the first time, which further confirms the energy competition between tumors and liver remnant due to the Warburg effect. Our results suggest that NEDC-assisted LDI MSI provides an in situ label-free analysis of multiple glycerophospholipids and low molecular weight metabolites (including glucose) with abundant peaks and high spatial resolution. This will allow future application to in situ definition of biomarkers, signaling pathways, and disease mechanisms.

1,5-Diaminonaphthalene hydrochloride assisted laser desorption/ionization mass spectrometry imaging of small molecules in tissues following focal cerebral ischemia.

A sensitive analytical technique for visualizing small endogenous molecules simultaneously is of great significance for clearly elucidating metabolic mechanisms during pathological progression. In the present study, 1,5-naphthalenediamine (1,5-DAN) hydrochloride was prepared for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of small molecules in liver, brain, and kidneys from mice. Furthermore, 1,5-DAN hydrochloride assisted LDI MSI of small molecules in brain tissue of rats subjected to middle cerebral artery occlusion (MCAO) was carried out to investigate the altered metabolic pathways and mechanisms underlying the development of ischemic brain damage. Our results suggested that the newly prepared matrix possessed brilliant features including low cost, strong ultraviolet absorption, high salt tolerance capacity, and fewer background signals especially in the low mass range (typically m/z < 500), which permitted us to visualize the spatial distribution of a broad range of small molecule metabolites including metal ions, amino acids, carboxylic acids, nucleotide derivatives, peptide, and lipids simultaneously. Nineteen endogenous metabolites involved in metabolic networks such as ATP metabolism, tricarboxylic acid (TCA) cycle, glutamate-glutamine cycle, and malate-aspartate shuttle, together with metal ions and phospholipids as well as antioxidants underwent relatively obvious changes after 24 h of MCAO. The results were highly consistent with the data obtained by MRM MS analysis. These findings highlighted the promising potential of the organic salt matrix for application in the field of biomedical research.