Xiaoyun Gong

Multicolor Imaging of Cancer Cells with Fluorophore-Tagged Aptamers for Single Cell Typing

The discrimination of the type of cancer cells remains challenging due to the subtle differences in their expression of membrane receptors. In this work, we developed a multicolor cell imaging method for distinguishing the type of cancer cells with fluorophore-tagged aptamers. We found that the interaction between aptamers and cancer cells was affected by both of the sequence of aptamers and the labeled dyes. As the co-ownership of biomarkers for different cancer cell lines, the fluorophore-tagged aptamers interacted with different cancer cell lines in different degree, resulting in a distinct color to discriminate the type of cancer cells at single cell level. Taking advantage of the cross-reactive ability of the fluorophore-tagged aptamers, we could not only distinguish the cancerous cells quickly from large quantities of noncancerous cells, but also identify the type of the cancerous cells. This work has potential application for cancer diagnostic and therapy in the future.

Rapid Removal of Matrices from Small-Volume Samples by Step-Voltage Nanoelectrospray†

Matrix unloaded: By changing from fixed-voltage (left) to step-voltage nanoelectrospray (right), the mass-spectrometric analysis of small-volume physiological samples is possible. Separation and ionization are achieved in one process, which avoids sample loss and dilution and prevents interference by the matrix. The result is high sensitivity even for samples at the nanoliter level.

Controlling Charge States of Peptides through Inductive Electrospray Ionization Mass Spectrometry

A novel ionization device for controlling the charge states of peptides based on an inductive elecrospray ionization technique was developed. This ion source keeps the major capabilities of electrospray ionization (ESI) which is compatible with liquid separation techniques (such as liquid chromatography (LC) and capillary electrophoresis (CE)) and can be potentially used to control the charge states of peptides accurately by simply varying the AC voltage applied. In comparison with conventional ESI, inductive ESI successfully simplifies the mass spectrum by reducing the charge states of peptide to a singly charged one, as well as eliminating the adduct ions.

Integrated Droplet-Based Microextraction with ESI-MS for Removal of Matrix Interference in Single-Cell Analysis

Integrating droplet-based microfluidics with mass spectrometry is essential to high-throughput and multiple analysis of single cells. Nevertheless, matrix effects such as the interference of culture medium and intracellular components influence the sensitivity and the accuracy of results in single-cell analysis. To resolve this problem, we developed a method that integrated droplet-based microextraction with single-cell mass spectrometry. Specific extraction solvent was used to selectively obtain intracellular components of interest and remove interference of other components. Using this method, UDP-Glc-NAc, GSH, GSSG, AMP, ADP and ATP were successfully detected in single MCF-7 cells. We also applied the method to study the change of unicellular metabolites in the biological process of dysfunctional oxidative phosphorylation. The method could not only realize matrix-free, selective and sensitive detection of metabolites in single cells, but also have the capability for reliable and high-throughput single-cell analysis.

Desalting by Crystallization: Detection of Attomole Biomolecules in Picoliter Buffers by Mass Spectrometry

Sensitive detection of biomolecules in small-volume samples by mass spectrometry is, in many cases, challenging because of the use of buffers to maintain the biological activities of proteins and cells. Here, we report a highly effective desalting method for picoliter samples. It was based on the spontaneous separation of biomolecules from salts during crystallization of the salts. After desalting, the biomolecules were deposited in the tip of the quartz pipet because of the evaporation of the solvent. Subsequent detection of the separated biomolecules was achieved using solvent assisted electric field induced desorption/ionization (SAEFIDI) coupled with mass spectrometry. It allowed for direct desorption/ionization of the biomolecules in situ from the tip of the pipet. The organic component in the assistant solvent inhibited the desorption/ionization of salts, thus assured successful detection of biomolecules. Proteins and peptides down to 50 amol were successfully detected using our method even if there were 3 × 10(5) folds more amount of salts in the sample. The concentration and ion species of the salts had little influence on the detection results.

Observation of Replacement of Carbon in Benzene with Nitrogen in a Low-Temperature Plasma

Selective activation of benzene has been mainly limited to the C-H activation. Simple replacement of one carbon in benzene with another atom remains unresolved due to the high dissociation energy. Herein, we demonstrate a direct breakage of the particularly strong C = C bond in benzene through ion-molecule reaction in a low-temperature plasma, in which one carbon atom was replaced by one atomic nitrogen with the formation of pyridine. The mechanism for the formation of pyridine from benzene has been proposed based on the extensive investigation with tandem mass spectrometry. The reaction pathway also works to other aromatics such as toluene and o-xylene. This finding provides a new avenue for selective conversion of aromatics into nitrogen-containing compounds.

Low-temperature plasma ionization source for the online detection of indoor volatile organic compounds

A simple-structure, low-power, and low-cost low temperature plasma (LTP) ionization source, coupled with mass spectrometry, for the online detection of indoor volatile organic compounds (VOCs) has been constructed in this work. Air, instead of noble gases, was employed as the discharging and carrier gas. And a custom-built AC high-voltage power supply with a total power consumption of 5 W, frequency of 2-4 kHz, and amplitude around 1-5 kV(p-p) was used. This LTP source is a soft ionization source. The initial performance of the ionization source has been evaluated by ionizing samples including alcohols, ketones, aldehydes and aromatics. These compounds cover most of the common air pollutants concerning peoples health. It is well known that those plasmas generated by dielectric barrier discharge (DBD) produce significant amount of metastable species and electrons with mean energies greater than several electronvolt, but minimal fragmentation was observed in our work. Protonated ions are the dominant product for the VOCs detected after the ionization process. Further work has been conducted to confirm the detection feature of this source. The results are promising enough to ensure the novel LTP ionization source as an effective tool for the online detection of indoor VOCs.

Hydrogen sulfide detection based on reflection: from a poison test approach of ancient China to single-cell accurate localization.

With the inspiration of an ancient Chinese poison test approach, we report a rapid hydrogen sulfide detection strategy in specific areas of live cells using silver needles with good spatial resolution of 2 × 2 μm(2). Besides the accurate-localization ability, this reflection-based strategy also has attractive merits of convenience and robust response when free pretreatment and short detection time are concerned. The success of endogenous H2S level evaluation in cellular cytoplasm and nuclear of human A549 cells promises the application potential of our strategy in scientific research and medical diagnosis.

Investigating the structural transitions of proteins during dissolution by mass spectrometry

An appropriate solvent environment is essential for the implementation of biological functions of proteins. Interactions between protein residues and solvent molecules are of great importance for proteins to maintain their active structure and catalyze biochemical reactions. In this study, we investigated such interactions and studied the structural transitions of proteins during their dissolution process. Our previously developed technique, namely solvent assisted electric field induced desorption/ionization, was used for the dissolution and immediate ionization of proteins. Different solvents and proteins were involved in the investigation. According to the results, cytochrome c underwent significant unfolding during dissolution in the most commonly used NH4Ac buffer. The unfolding got more serious when the concentration of NH4Ac was further increased. Extending the dissolution time resulted in the re-folding of cytochrome c. In comparison, no unfolding was observed if cytochrome c was pre-dissolved in NH4Ac buffer and detected by nano-ESI. Furthermore, no unfolding was observed during the dissolution process of cytochrome c in water. Interactions between the residues of cytochrome c and the solute of NH4Ac might be the reason for the unfolding phenomenon. Similar unfolding phenomenon was observed on holo-myoglobin. However, the observed dissolution feature of insulin was different. No unfolding was observed on insulin during dissolution in NH4Ac buffers. Insulin underwent observable unfolding when water was used for dissolution. This might be due to the structural difference between different proteins. The obtained results in the present study furthered our insights into the interactions between proteins and the solvents during the phase transition of dissolution.

Boosting the Signal Intensity of Nanoelectrospray Ionization by Using a Polarity-Reversing High-Voltage Strategy

Continuous efforts have been made to further improve the performance of nano-ESI. In this work, we made use of a polarity-reversing high-voltage strategy for the generation of nano-ESI (PR-nESI). Typically, a negative high voltage of -3.0 kV was first applied to the electrode and maintained for 6 s. Then the polarity was reversed, and a positive high voltage of +1.75 kV was applied for the generation of electrospray. Compared with conventional nano-ESI, PR-nESI significantly enhanced the signal intensity of protonated protein ions. The signal-to-noise ratio (S/N) of protonated protein ions was increased by 1-2 orders of magnitude. The increase of S/N was even more remarkable at lower concentrations. Furthermore, PR-nESI also had a desalting effect. Metal ion adducts of proteins were effectively removed. No metal ion adducts were identified from the spectra, even if the concentration of salt was increased to the millimolar level. The performance of PR-nESI was confirmed in the detection of different molecules including proteins, peptides, amino acids, and other small-molecule compounds. The intact folded structure of proteins was preserved during PR-nESI. No unfolding was observed in the spectra. PR-nESI was further applied to the analysis of noncovalent protein-ligand complexes and protein digest. At last, investigations into the mechanism of PR-nESI were carried out. The enhancement of signal intensity and desalting effect were related to the electromigration of the solutes in solution. With all the advantages above, PR-nESI would be a promising method in the future analytical and bioanalytical applications.