Temperature-programmed native electrospray ionization mass spectrometry gives detailed insight into the assembly of model collagen triple helices.

Single-cell metabolomics is expected to deliver fast and dynamic information on cell function; therefore, it requires rapid analysis of a wide variety of very small quantities of metabolites in living cells. In this work, a hybrid ionization source that combines nanoelectrospray ionization (nanoESI) and dielectric barrier discharge ionization (DBDI) is proposed for single-cell analysis. A capillary with a 1 μm i.d. tip was inserted into cells for sampling and then directly used as the nanoESI source for ionization of polar metabolites. In addition, a DBDI source was employed as a post-ionization source to improve the ionization of apolar metabolites in cells that are not easily ionized by ESI. By increasing the voltage of the DBDI source from 0 to 3.2 kV, the classes of detected metabolites can be shifted from mostly polar to both polar and apolar to mainly apolar. Plant cells (onion) and human cells (PANC-1) were investigated in this study. After optimization, 50 compounds in onion cells and 40 compounds in PANC-1 cells were observed in ESI mode (3.5 kV) and an additional 49 compounds in onion cells and 73 compounds in PANC-1 cells were detected in ESI (3.5 kV)-DBDI (2.6 kV) hybrid mode. This hybrid ionization source improves the coverage, ionization efficiency, and limit of detection of metabolites with different polarities and could potentially contribute to the fast-growing field of single-cell metabolomics.

The direct formation of N-heterocycles from aromatic hydrocarbons has been observed in nitrogen-based lowtemperature plasmas; the mechanism of this unusual nitrogenfixation reaction is the topic of this paper. We used homologous aromatic compounds to study their reaction with reactive nitrogen species (RNS) in a dielectric barrier discharge ionization (DBDI) source. Toluene (C 7 H 8 ) served as a model compound to study the reaction in detail, which leads to the formation of two major products at "high" plasma voltage: a nitrogen-replacement product yielding protonated methylpyridine (C 6 H 8 N + ) and a protonated nitrogen-addition (C 7 H 8 N + ) product. We complemented those studies by a series of experiments probing the potential mechanism. Using a series of selected-ion flow tube experiments, we found that N + , N 2 + , and N 4 + react with toluene to form a small abundance of the N-addition product, while N( 4 S) reacted with toluene cations to form a fragment ion. We created a model for the RNS in the plasma using variable electron and neutral density attachment mass spectrometry in a flowing afterglow Langmuir probe apparatus. These experiments suggested that excited-state nitrogen atoms could be responsible for the N-replacement product. Density functional theory calculations confirmed that the reaction of excitedstate nitrogen N( 2 P) and N( 2 D) with toluene ions can directly form protonated methylpyridine, ejecting a carbon atom from the aromatic ring. N( 2 P) is responsible for this reaction in our DBDI source as it has a sufficient lifetime in the plasma and was detected by optical emission spectroscopy measurements, showing an increasing intensity of N( 2 P) with increasing voltage.

We tested a paper-based platform ("Aptapaper") for the upconcentration and analysis of small molecules from complex matrices for two well-characterized aptamers, quinine and serotonin binding aptamers (QBA and SBA, respectively). After incubating the aptapaper under conditions that ensure correct aptamer folding, the aptapaper was used to upconcentrate target analytes from complex matrices. Aptapaper was rinsed, dried, and the target analyte was detected immediately or up to 4 days later by paper spray ionization coupled to high-resolution mass spectrometry (PS-MS). The minimum concentrations detectable were 81 pg/mL and 1.8 ng/mL for quinine and serotonin, respectively, from 100 mM AmAc or water. Complementary characterization of the QBA aptapaper system was performed using an orthogonal fluorescence microscopy method. Random adsorption was analyte-specific and observed for quinine, but not serotonin. This aptapaper approach is a semiquantitative (10−20% RSD) platform for upconcentration of small metabolites by mass spectrometry.

Soft ambient ionization sources generate reactive species that interact with analyte molecules to form intact molecular ions, which allows rapid, sensitive, and direct identification of the molecular mass. We used a dielectric barrier discharge ionization (DBDI) source with nitrogen at atmospheric pressure to detect alkylated aromatic hydrocarbon isomers (C 8 H 10 or C 9 H 12 ). Intact molecular ions [M] •+ were detected at 2.4 kV pp , but at increased voltage (3.4 kV pp ), [M + N] + ions were formed, which could be used to differentiate regioisomers by collision-induced dissociation (CID). At 2.4 kV pp , alkylbenzene isomers with different alkyl-substituents could be identified by additional product ions: ethylbenzene and -toluene formed [M-2H] + , isopropylbenzene formed abundant [M-H] + , and propylbenzene formed abundant C 7 H 7 + . At an operating voltage of 3.4 kV pp , fragmentation of [M + N] + by CID led to neutral loss of HCN and CH 3 CN, which corresponded to steric hindrance for excited state N-atoms approaching the aromatic ring (C-H). The ratio of HCN to CH 3 N loss (interday relative standard deviation [RSD] < 20%) was distinct for ethylbenzene and ethyltoluene isomers. The greater the number of alkyl-substituents (C-CH 3 ) and the more sterically hindered (meta > para > ortho) the aromatic core, the greater the loss of CH 3 CN relative to HCN was.