CBT-Cys click condensation reaction has a high second-order reaction rate constant and has found wide applicability in recent years. However, its reaction mechanism has not been experimentally validated and its application for identifying bioactive N-terminal Cys peptides in real clinical samples has not been reported. Herein, firstly, by employing induced nanoelectrospray ionization-mass spectrometry (InESI-MS) and a home-built micro-reactor, we successfully intercepted and structurally characterized the crucial intermediate in this click reaction for the first time. With the intermediate, the proposed mechanism of this reaction was corroborated. Moreover, we also applied this MS setup to monitor the reaction in real time and obtained the second-order reaction rate constants of this reaction at different pH values. After mechanistic study, we applied this click reaction for identifying bioactive N-terminal cysteine peptides in amniotic fluid (AF). Eight unique N-terminal Cys peptides in AF, three of which are located in the functional domain regions of their corresponding proteins, were identified with a false positive rate less than 1%. One of the three peptides was found able to inhibit the growth of uterine endometrial cancer HEC-1-B cells but not the endometrial normal cells via a typical apoptotic pathway. With its mechanism satisfactorily elucidated, the kinetic parameters obtained, as well as its application for fishing bioactive N-terminal Cys peptides from vast complex clinical samples, we anticipate that this CBT-Cys click reaction could be applied more widely for the facile isolation, site-specific identification, and quantification of N-terminal Cys-containing peptides in complex biological samples.

•ZrO2@paper was firstly used for selective capture of ribonucleosides.•Interferences from amines could be excluded under basic environment.•ZrO2@paper extraction – MS provides a rapid and facile quantification strategy.•This method is potentially useful for developing rapid and low-cost diagnostics.A rapid and facile analytical method for quantification of ribonucleosides in human urine was developed by the combination of nanocoating cellulose paper based microextraction and nanoelectrospray ionization-tandem mass spectrometry (nESI-MS/MS). Cellulose paper used for microextraction was modified by nano-precision deposition of uniform ultrathin zirconia gel film using a sol-gel process. Due to the large surface area of the cellulose paper and the strong affinity between zirconia and the cis-diol compounds, the target analytes were selectively extracted from the complex matrix. Thus, the detection sensitivity was greatly improved. Typically, the nanocoating cellulose paper was immersed into the diluted urine for selective extraction of target analytes, then the extracted analytes were subjected to nESI-MS/MS detection. The whole analytical procedure could be completed within 10 min. The method was evaluated by the determination of ribonucleosides (adenosine, cytidine, uridine, guanosine) in urine sample. The signal intensities of the ribonuclesides extracted by the nanocoating cellulose paper were greatly enhanced by 136–459-folds compared with the one of the unmodified cellulose paper based microextraction. The limits of detection (LODs) and the limits of quantification (LOQs) of the four ribonucleosides were in the range of 0.0136–1.258 μg L−1 and 0.0454–4.194 μg L−1, respectively. The recoveries of the target nucleosides from spiked human urine were in the range of 75.64–103.49% with the relative standard deviations (RSDs) less than 9.36%. The results demonstrate the potential of the proposed method for rapid and facile determination of endogenous ribonucleosides in urine sample.Ribonucleosides could be selectively captured by the ZrO2@paper from human urine and then direct quantified by nESI-MS/MS within 10 min.Download high-res image (193KB)Download full-size image

•Water molecules not NaBH4 are the hydrogen source for the reduced amino groups.•NaBH4 could contribute to the ionization of H+ from water.•This reduction process is spontaneous to some extent.New insights for the mechanism of nitro to amino groups conversion have been revealed on both N-doped graphene and Ag nanoparticles catalysts, based on the paper assisted ultrasonic spray ionization mass spectrometry: (1) water molecules not NaBH4 are the hydrogen source for the reduced amino groups, (2) NaBH4 could contribute to the ionization of H+ from water, facilitating its adsorption on nitro groups, (3) six different intermediates have been detected to depict the whole catalytic process, and no condensed roots are involved in and (4) this reduction process is spontaneous to some extent, and even without catalysts it is not totally stopped as obtained from the spectral measurements.Download high-res image (38KB)Download full-size image

The identification of endogenous proteins as well as their binding to metal ions in living cells is determined by combining pulsed electrophoretic separations with nanoelectrospray ionization followed by mass spectrometric detection. This approach avoids problems resulting from the complicated cellular environment. In this manner, we demonstrate the rapid identification (300 ms or less) of intact proteins from living E. coli cells including the complexation of calmodulin with calcium ion. The latter showed different binding states from those observed in in vitro studies. These observations also reveal in vitro measurements do not necessarily represent the actual situation in living cells. We conclude that the attempted in situ measurement of intracellular proteins with minimal sampling processes should be preferred.

Accurate mass spectrometry (MS) signal for peptide/protein analysis, which could be affected by various MS conditions, plays an essential role in identification and quantification of biological samples. Herein, we tried to alleviate the possible interferences from electrochemical oxidations during electrospray ionization (ESI). Three most common electrochemical oxidation reactions in ESI include oxidation of analyte, solvent, and electrode. With introduction of induced electrospray ionization (IESI) (a variant form of ESI), these interferences were significantly alleviated for peptides/proteins. That effect was also tested with flow injection experiments with different solution flow rates, electrolyte concentrations and solvent compositions, which was to simulate various chromatography conditions in conventional liquid chromatography (LC) separations. For all chromatography conditions tested, electrochemical oxidation was significantly alleviated for the absence of physical contact between spray solution and electrode.

•A humidity independent mass spectrometric method for gas phase samples analysis.•A universal and good sensitivity method.•The method can real time identify plant released raw chemicals.In this work, a humidity independent mass spectrometric method was developed for rapid analysis of gas phase chemicals. This method is based upon ambient proton transfer reaction between gas phase chemicals and charged water droplets, in a reaction chamber with nearly saturate humidity under atmospheric pressure. The humidity independent nature enables direct and rapid analysis of raw gas phase samples, avoiding time- and sample-consuming sample pretreatments in conventional mass spectrometry methods to control sample humidity. Acetone, benzene, toluene, ethylbenzene and meta-xylene were used to evaluate the analytical performance of present method. The limits of detection for benzene, toluene, ethylbenzene and meta-xylene are in the range of ∼0.1 to ∼0.3 ppbV; that of benzene is well below the present European Union permissible exposure limit for benzene vapor (5 μg m−3, ∼1.44 ppbV), with linear ranges of approximately two orders of magnitude. The majority of the homemade device contains a stainless steel tube as reaction chamber and an ultrasonic humidifier as the source of charged water droplets, which makes this cheap device easy to assemble and facile to operate. In addition, potential application of this method was illustrated by the real time identification of raw gas phase chemicals released from plants at different physiological stages.Direct and humidity independent mass spectrometry analysis of gas phase chemicals could be achieved via ambient proton transfer ionization, ion intensity was found to be stable with humidity ranged from ∼10% to ∼100%.

Dissociation of disulfide is normally mandatory prior to disulfide peptide sequencing via electrospray ionization collision induced dissociation mass spectrometry (ESI-CID-MS). Herein, a facile method for directly sequencing intact disulfide peptides was proposed. The basic principles involved were electrolyte-enhanced corona discharge in ESI and the following oxidative cleavage reaction.

Herein, we developed an in situ disulfide cleavage protocol. It is based on the selective reaction between disulfide and the hydroxyl radical, which could be rapidly and controllably generated via induced nanoelectrospray. In less than one minute, increased disulfide peptide sequence coverage could be obtained via switching the “cleavage ON/OFF” modes between conventional and induced nanoelectrospray.

To analyze compounds in complicated matrixes using mass spectrometry, we describe a novel ambient ionization approach, termed paper assisted ultrasonic spray ionization (PAUSI). The ionization process is based on the ultrasonic vibration of the piezoelectric ceramic disk, on which the samples are placed. Porous materials are utilized to generate fine initial droplet, which could alleviate matrix effect during ionization process for complicated matrix. PAUSI was evaluated as an attractive tool to screen analytes from complicated matrixes, such as (1) bovine serum with NaCl 150 g/L, (2) viscous samples, and (3) biological fluid, without any sample preparation. Moreover, it provides great advantage in simplifying the mass spectrometry analysis process, and the ionization device is inexpensive and easy to operate.

CBT-Cys click condensation reaction has a high second-order reaction rate constant and has found wide applicability in recent years. However, its reaction mechanism has not been experimentally validated and its application for identifying bioactive N-terminal Cys peptides in real clinical samples has not been reported. Herein, firstly, by employing induced nanoelectrospray ionization-mass spectrometry (InESI-MS) and a home-built micro-reactor, we successfully intercepted and structurally characterized the crucial intermediate in this click reaction for the first time. With the intermediate, the proposed mechanism of this reaction was corroborated. Moreover, we also applied this MS setup to monitor the reaction in real time and obtained the second-order reaction rate constants of this reaction at different pH values. After mechanistic study, we applied this click reaction for identifying bioactive N-terminal cysteine peptides in amniotic fluid (AF). Eight unique N-terminal Cys peptides in AF, three of which are located in the functional domain regions of their corresponding proteins, were identified with a false positive rate less than 1%. One of the three peptides was found able to inhibit the growth of uterine endometrial cancer HEC-1-B cells but not the endometrial normal cells via a typical apoptotic pathway. With its mechanism satisfactorily elucidated, the kinetic parameters obtained, as well as its application for fishing bioactive N-terminal Cys peptides from vast complex clinical samples, we anticipate that this CBT-Cys click reaction could be applied more widely for the facile isolation, site-specific identification, and quantification of N-terminal Cys-containing peptides in complex biological samples.