Nanoliter atmospheric pressure photoionization-mass spectrometry for direct bioanalysis of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are a class of low-polarity environmental contaminants that have severe carcinogenic effects and have drawn worldwide attention. However, there remain challenges for current mass spectrometric ionization techniques in the analysis of low-polarity compounds in small-volume biosamples, such as single cells. In this work, we developed a nanoliter atmospheric pressure photoionization (nano-APPI) source and optimized its parameters for the detection of PAHs in small-volume samples. We evaluated the ionization performance of the source in direct and auxiliary gas-assisted photoionization modes and analyzed different PAH compounds as well as spiked biosamples. By combining the advantages of nano-electrospray ionization (nano-ESI) and atmospheric pressure photoionization (APPI), our newly developed nano-APPI source achieved high sensitivity for the analysis of PAHs down to the fmol level. Compared to conventional atmospheric pressure chemical ionization (APCI), the detection limit of PAHs was increased by 1-2 orders of magnitude. By optimizing various parameters, we achieved highly efficient ionization of PAHs, effective analysis of PAHs in mixed components, and sensitive detection of low-abundance PAHs in single-cell samples. Our optimized nano-APPI source was successfully applied for the sensitive analysis of PAHs in complex biological samples. Based on our study, we believe that nano-APPI holds great promise for toxicological studies on complex biological samples. The present work has implications for improving the detection sensitivity of low-polarity environmental contaminants and advancing the field of MS-based analysis of small-volume biosamples.

Investigation on acquired palbociclib resistance by LC-MS based multi-omics analysis.

Palbociclib is a specific CDK4/6 inhibitor that has been widely applied in multiple types of tumors. Different from cytotoxic drugs, the anticancer mechanism of palbociclib mainly depends on cell cycle inhibition. Therefore, the resistance mechanism is different. For clinical cancer patients, drug resistance is inevitable for almost all cancer therapies including palbociclib. We have trained palbociclib resistant cells in vitro to simulate the clinical situation and applied LC-MS multi-omics analysis methods including proteomic, metabolomic, and glycoproteomic techniques, to deeply understand the underly mechanism behind the resistance. As a result of proteomic analysis, the resistant cells were found to rely on altered metabolic pathways to keep proliferation. Metabolic processes related to carbohydrates, lipids, DNA, cellular proteins, glucose, and amino acids were observed to be upregulated. Most dramatically, the protein expressions of COX-1 and NDUFB8 have been detected to be significantly overexpressed by proteomic analysis. When a COX-1 inhibitor was hired to combine with palbociclib, a synergistic effect could be obtained, suggesting the altered COX-1 involved metabolic pathway is an important reason for the acquired palbociclib resistance. The KEGG pathway of N-glycan biosynthesis was identified through metabolomics analysis. N-glycoproteomic analysis was therefore included and the global glycosylation was found to be elevated in the palbociclib-resistant cells. Moreover, integration analysis of glycoproteomic data allowed us to detect a lot more proteins that have been glycosylated with low abundances, these proteins were considered to be overwhelmed by those highly abundant proteins during regular proteomic LC-MS detection. These low-abundant proteins are mainly involved in the cellular biology processes of cell migration, the regulation of chemotaxis, as well as the glycoprotein metabolic process which offered us great more details on the roles played by N-glycosylation in drug resistance. Our result also verified that N-glycosylation inhibitors could enhance the cell growth inhibition of palbociclib in resistant cells. The high efficiency of the integrated multi-omics analysis workflow in discovering drug resistance mechanisms paves a new way for drug development. With a clear understanding of the resistance mechanism, new drug targets and drug combinations could be designed to resensitize the resistant tumors.

Analysis of low-abundance molecules in complex matrices by quadrupole-linear ion trap mass spectrometry using a simultaneous fragmentation and accumulation strategy.

RATIONALE: Fast and sensitive analysis of low-abundance molecules in complex matrices has always been a challenge in chemical and biological applications. Mass spectrometry (MS) has been widely used in the fields of chemical and biological analysis due to its unparalleled specificity and sensitivity. However, the MS signals consistently deteriorate in the presence of matrices. Demands for more sensitive and efficient methods to analyze those low-abundance molecules in chemical and biological systems are in urgent need. METHODS: Based on a home-made quadrupole-linear ion trap (Q-LIT) mass spectrometer, a simultaneous fragmentation and accumulation strategy was developed to improve the sensitivity of the analysis for the low-abundance molecules in complex matrices. Ions were filtered by the quadrupole into the LIT. The precursor ions were fragmented and the product ions were isolated and accumulated in the LIT simultaneously. The fragmentation, isolation and accumulation processes were conducted at the same time. The accumulation time could be controlled to accumulate sufficient product ions. RESULTS: With this strategy, the signal intensity of targeted molecules could be increased by 2-8 times and by increasing the accumulation time, this could be further enhanced. Those interferences induced by isomers and matrices can be reduced by using our method. We further applied our method to the quantification and analysis of biological samples. Tryptic digested peptides of myoglobin (Mb) were successfully detected by our method. CONCLUSIONS: We have established a new method with great advantages in the detection of molecules in complex matrices. The application of this method promises better results in the bioanalytical area, especially for the analysis of substances in complex matrices in the future.

Direct analysis of hydroxylated polycyclic aromatic hydrocarbons in biological samples with complex matrices using polarity-reversed nanoelectrospray ionization.

RATIONALE: Polycyclic aromatic hydrocarbons (PAHs) are a class of environmental contaminants with carcinogenic effect drawing worldwide attention. PAHs can be converted into hydroxylated PAHs (OH-PAHs) through metabolic processes. Thus, they are commonly considered as an important class of biomarkers of PAH exposure. However, direct analysis of related metabolites of these environmental pollutants in biological samples using mass spectrometry is still challenging because of matrix effect and ion suppression during nanoelectrospray ionization (nano-ESI). METHODS: In our previous work, a polarity-reversed nanoelectrospray ionization (PR-nESI) technique was developed for the analysis of biomolecules in complex matrices. In this work, we further optimized PR-nESI for direct and sensitive analysis of OH-PAHs in different samples under severe salt interference in negative polarity. RESULTS: Compared with conventional nano-ESI, the optimized PR-nESI method realized sensitive detection of 1-naphthol in samples with a concentration of salt up to millimolar level. The signal-to-noise ratio (S/N) of OH-PAHs was increased by 1-2 orders of magnitude compared with conventional nano-ESI. Six different OH-PAHs were successfully detected with high S/N ratio using PR-nESI. PR-nESI was further successfully applied in the analysis of OH-PAHs in spiked fetal blood serum, human urine, and single-cell samples. For environmentally exposed subjects, the detections of OH-PAHs in single-cell samples and urines from human smokers were successfully conducted. CONCLUSION: The optimized PR-nESI method was successfully applied for the sensitive analysis of OH-PAHs in complex biological samples with severe salt effects. Based on the present study, PR-nESI can have a promising prospect for the sensitive analysis of other metabolites of environmental pollutants in negative polarity.

Intramolecular benzyl cation transfer in the gas-phase fragmentation of protonated benzyl phenyl sulfones.

In this study, the gas-phase fragmentations of protonated benzyl phenyl sulfones were investigated by electrospray ionization tandem mass spectrometry (ESI-MSn ). Upon collisional activation, several characteristic fragment ions were observed, and the similar results occurred with different substituted benzyl phenyl sulfones. A mechanism involving an intramolecular benzyl cation transfer and the formation of intermediate ion was proposed and further identified by density functional theory (DFT) calculations. In addition, a reference compound, benzenesulfinic acid benzyl ester, has been synthesized, and its protonated ion has the same gas-phase behavior as compared to the protonated benzyl phenyl sulfone. This work provides access to some insight into the intramolecular benzyl-transfer reactions of benzyl phenyl sulfones in the gas phase and orients the characteristic peaks in collision-induced dissociation spectrometry (CID-MS).

Spiropyran in Situ Switching: A Real-Time Fluorescence Strategy for Tracking DNA G-Quadruplexes in Live Cells.

DNA G-quadruplexes (G4s) in vivo have been linked to cancer and other diseases such as neurological disorders. Nondestructive fast detection of endogenous DNA G4s can provide specific real-time information, which is of particular interest for clinic accurate diagnosis. However, tools to probe live-cell endogenous DNA G4s in real time are very limited. Herein, we report the design and development of a fluorescent molecule QIN for the real-time detection of endogenous DNA G4s in live cells with the aid of a new spiropyran in situ switching (SIS) strategy. The lipophilic spiropyran-linked QIN differs from the other probes in that it can enter live cells readily within 15 s and can be in situ induced by DNA G4s to adopt its charged open form, causing a large red shift in the fluorescent emission wavelength. Live-cell super-resolution fluorescent imaging suggests that the SIS-based probe has high photostability and can be applied for the accurate detection of DNA G4s in complex biosystems with very high sensitivity and selectivity.

Mass spectrometric studies on the interaction of cisplatin and insulin.

The interaction of antitumor drug, cisplatin (cis-[PtCl2(NH3)2], CDDP) with insulin from porcine pancreas has been investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and high resolution hybrid ion trap/time-of-flight mass spectrometry (MALIDI-TOF/TOF-MS and ESI-IT/TOF MS). The MALDI-TOF/TOF-MS results demonstrated that the presence of cisplatin complex resulted in the reduction of the disulfide bond in porcine pancreas after the incubations of the two substances were performed in vitro. It indicated that the presence of cisplatin would destroy the native configuration of insulin, which may lead to the inactivation of insulin. High resolution mass values and the characteristic isotopic pattern of the platinated insulin ions allowed the analysis of platinated mono-, di- and triadducts of cisplatin and insulin in the incubations under different conditions. The laser-induced dissociation of the monoadduct obtained in MALDI source was carried out and one platinum was found to bind to insulin B chain was determined. The platinum binding sites were further identified to be the N terminus (B chain), cysteine 7 (B chain) and cysteine 19 (B chain) residues by electrospray ionization tandem mass spectrometry. The identification of the interaction between insulin and cisplatin broadens the horizon of the knowledge in the interaction of the proteins and metallodrugs.