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Sensitive analysis of metabolites in a single cell is of fundamental significance for the better understanding of biological variability, differential susceptibility in disease therapy, and cell-to-cell heterogeneity as well. Herein, polarity-specific profiling of metabolites in a single cell was implemented by probe electrophoresis mass spectrometry (PEMS), which combined electrophoresis sampling of metabolites from a single cell and nanoelectrospray ionization-mass spectrometry (nanoESI-MS) analysis of the sampled metabolites. Enhanced extraction of either negatively or positively charged metabolites from a single cell was achieved by applying a DC voltage offset of +2.0 and -2.0 V to the probe, respectively. The experimental data demonstrated that PEMS features high throughput (≥200 peaks) and high sensitivity (≥10-times signal enhancement for [Choline + H]

Traditionally, molecular information on metabolites, lipids, and proteins is collected from separate individual tissue samples using different analytical approaches. Herein a novel strategy to minimize the potential material losses and the mismatch between metabolomics, lipidomics, and proteomics data has been demonstrated based on internal extractive electrospray ionization mass spectrometry (iEESI-MS). Sequential detection of lipids, metabolites, and proteins from the same tissue sample was achieved without sample reloading and hardware alteration to MS instrument by sequentially using extraction solutions with different chemical compositions. With respect to the individual compound class analysis, the sensitivity, specificity, and accuracy obtained with the integrative information on metabolites, lipids, and proteins from 57 samples of 13 patients for lung cancer prediction was substantially improved from 54.0%, 51.0%, and 76.0% to 100.0%, respectively. The established method is featured by low sample consumption (ca. 2.0 mg) and easy operation, which is important to minimize systematic errors in precision molecular diagnosis and systems biology studies.

Micro-electrochemical probe mass spectrometry (μECP-MS) is demonstrated as a method for the direct profiling of chemical composition of bulk alloy samples without tedious sample pretreatment. The spatial distribution of Zn and Cu components of a Cu/Zn alloy sample was successively identified by scanning the electrolysis potential from -0.6 V to 0.6 V. The lateral resolution of alloy chemical profiling was ≤10 μm, and the depth resolution was ≤0.5 nm. Besides metal components, the method also allows the simultaneous detection of organic molecules on the sample surface. The limit of detection for Rhodamine B, Zn, and Cu depositions was 4.47, 9.58, and 24.25 ag per μm2, respectively. The method is particularly useful for high-throughput (

Supercharged protein ions produced by electrospray ionization are extremely efficient proton donors for secondary ionization. Here, by electrospraying the protein solutions containing 5% 1,2-butylene carbonate, the supercharged protein ions with unusually high proton density were produced as the primary ions for the ionization of exhaled breath samples in the extractive electrospray ionization mass spectrometry (EESI-MS), which resulted in the enhanced ionization efficiency for the breath analytes even with relatively low gas phase basicity. Moreover, the total number of metabolites detected in breath increased by about 260% in the mass range of 200-500 Da, owing to the substantial signal enhancement for breath metabolites, providing complementary and additional information to conventional SESI.

Rapid analysis of phosphopeptides and phospholipids in biological matrixes is of significant interest in multiple disciplines of life sciences. Herein, trace phospholipids in human plasma, whole blood, and undiluted human urine as well as phosphopeptides in protein digest were selectively captured on a homemade array of TiO2 nanowires for sensitive characterization by internal extractive electrospray ionization mass spectrometry (TiO2-iEESI-MS). Sequential release of captured chemicals from TiO2 array was achieved by tuning pH of the extraction solvent. A single sample analysis, including sample loading, chemical extraction and MS detection, was accomplished within 3 min. As far as the quantification of phospholipids, acceptable linearity (R2 ≥ 0.9985) and relative standard deviations (RSDs ≤ 8.9%) were obtained within the range of 0.1–500 μg L–1 for LysoPC(14:0) and LysoPC(16:0) in raw urine samples. Limit of detection (LOD) ≤ 0.025 μg L–1 and recovery rates of 94.8–101.6% were obtained for these phospho...

Rapid chemical decoding of bulk alloys to obtain both organic and elemental composition is of sustainable interest in multiple disciplines. Herein, an analytical strategy inherited from electrochemistry and mass spectrometry (MS) was developed for direct molecular characterization of alloys. While the organics on the alloy surface were simply extracted into the solvent for ESI-MS analysis, the components in the bulk alloy were successively converted into metal ions at appropriate electrolysis potentials, which were online chelated with specific ligands for ESI-MS analysis. A single sample analysis took only a few seconds since no other sample pretreatment was required, and a detection limit of 0.1 ppb was achieved for a component in alloy with low sample consumption (1.0 mg). Proof-of-concept application indicated that the presented method has unique capability for successive analysis of organic and metallic components in liquids (e.g., engine oil), solids (e.g., alloy), and tunable spatial resolution (∼1.0 to (1.0 × 10

Significant efforts are being made to develop more practical and versatile approaches for the preconcentration and purification of complex chemical samples. Inspired by the naturally occurring enrichment of organic compounds in sea aerosols, in this study we demonstrate the potential of induced bubble bursting as an approach for the preconcentration of organic solutes in various aqueous solutions. Apart from the preconcentration of organics, notable decrease in the concentration of metal salt components was discovered for the first time. On the basis of a series of model experiments, the phenomenon has been attributed to intermolecular competition at the surface interface of rising bubbles. Overall, our results indicate the high versatility and simplicity of the bubble bursting approach for the simultaneous preconcentration and desalting of organic solutes in aqueous solutions for mass spectrometry, chromatography, optical detection, and other fields of analysis.

Rapid analysis of phosphopeptides and phospholipids in biological matrixes is of significant interest in multiple disciplines of life sciences. Herein, trace phospholipids in human plasma, whole blood, and undiluted human urine as well as phosphopeptides in protein digest were selectively captured on a homemade array of TiO

Ionization of proteins and noncovalent protein complexes with minimal disturbance to their native structure presents a great challenge for biological mass spectrometry (MS). In living organisms, the native structure of intracellular proteins is commonly stabilized by solute amino acids (AAs) accumulated in cells at very high concentrations. Inspired by nature, we hypothesized that AAs could also pose a stabilizing effect on the native structure of proteins and noncovalent protein complexes during ionization. To test this hypothesis, here we explored MS response for various protein complexes upon the addition of free AAs at mM concentrations into the electrospray ionization (ESI) solution. Thermal activation of ESI droplets in the MS inlet capillary was employed as a model destabilizing factor during ionization. Our results indicate that certain AAs, in particular proline (Pro), pose considerable positive effect on the stability of noncovalent protein complexes in ESI-MS without affecting the signal intensity of protein ions and original protein-ligand equilibrium, even when added at the 20 mM concentration. The data suggest that the degree of protein stabilization is primarily determined by the osmolytic and ampholytic characteristics of AA solutes. The highest stability and visibility of noncovalent protein complexes in ESI-MS are achieved using AA additives with neutral isoelectric point, moderate proton affinity, and unfavorable interaction with the native protein state. Overall, our results indicate that the simple addition of free amino acids into the working solution can notably improve the stability and accuracy of protein analysis by native ESI-MS.

We introduce an online multiple-junction capillary isoelectric focusing fractionator (OMJ-CIEF) for separation of biological molecules in solution by pI. In OMJ-CIEF, the separation capillary is divided into seven equal sections joined with each other via tubular Nafion membrane insertions. Each junction is communicated with its own external electrolytic buffer which is used both to supply electrical contact and for solvent exchange. The performance of the fractionator was explored using protein and peptide samples covering broad pI range. Separation was achieved in ionic and ampholytic buffers, including ammonium formate, ammonium hydroxide, histidine, and arginine. By maintaining electric potential across upstream segments of the capillary after the focusing stage, selective release of downstream analyte fractions could be achieved. The selective release mode circumvents the problem of peak broadening during mobilization and enables convenient comprehensive sampling for orthogonal separation methods. Using single-component ampholyte buffers with well-defined pI cutoff values, controlled separation of protein mixture into basic and acidic fractions was demonstrated. The device is cheap and easy to fabricate in-house, simple in operation, and straightforward in interfacing to hyphened analytical platforms. OMJ-CIEF has a potential of becoming a practical add-on unit in a wide range of bioanalytical setups, in particular as a first-dimension separation in mass spectrometry based proteomics or as a preparative tool for analyte purification, fractionation, and preconcentration.

The powerful hybrid analysis method of capillary-based separations followed by mass spectrometric analysis gives substantial chemical identity and structural information. It is usually carried out using electrospray ionization. However, the salts and detergents used in the mobile phase for electrokinetic separations suppress ionization efficiencies and contaminate the inlet of the mass spectrometer. This report describes a new method that uses desorption electrospray ionization (DESI) to overcome these limitations. Effluent from capillary columns is deposited on a rotating Teflon disk that is covered with paper. As the surface rotates, the temporal separation of the eluting analytes (i.e., the electropherogram) is spatially encoded on the surface. Then, using DESI, surface-deposited analytes are preferentially ionized, reducing the effects of ion suppression and inlet contamination on signal. With the use of this novel approach, two capillary-based separations were performed: a mixture of the rhodamine dyes at milligram/milliliter levels in a 10 mM sodium borate solution was separated by capillary electrophoresis, and a mixture of three cardiac drugs at milligram/milliliter levels in a 12.5 mM sodium borate and 12.5 mM sodium dodecyl sulfate solution was separated by micellar electrokinetic chromatography. In both experiments, the negative effects of detergents and salts on the MS analyses were minimized.

Characterization of enzymatic reactions occurring in untreated biological samples is of increasing interest. Herein, the chemical conversion of alliin to allicin, catalyzed by allinase, in raw garlic cloves has been followed in vivo by internal extractive electrospray ionization mass spectrometry (iEESI-MS). Both precursors and products of the enzymatic reaction were instantaneously extracted by infused solution running throughout the tissue and directly electrospray ionized on the edge of the bulk sample for online MS analysis. Compared to the room-temperature (+25 °C) scenario, the alliin conversion in garlic cloves decreased by (7.2 ± 1.4) times upon heating to +80 °C and by (5.9 ± 0.8) times upon cooling to -16 °C. Exposure of garlic to gentle ultrasound irradiation for 3 h accelerated the reaction by (1.2 ± 0.1) times. A 10 s microwave irradiation promoted alliin conversion by (1.6 ± 0.4) times, but longer exposure to microwave irradiation (90 s) slowed the reaction by (28.5 ± 7.5) times compared to the reference analysis. This method has been further employed to monitor the germination process of garlic. These data revealed that over a 2 day garlic sprouting, the allicin/alliin ratio increased by (2.2 ± 0.5) times, and the averaged degree of polymerization for the detected oligosaccharides/polysaccharides decreased from 11.6 to 9.4. Overall, these findings suggest the potential use of iEESI-MS for in vivo studies of enzymatic reactions in native biological matrices.

Recently, we introduced an online multijunction capillary isoelectric focusing (OMJ-CIEF) fractionator to fractionate proteins and peptides in electrospray-friendly solution. In this follow-up study, the original configuration of the fractionator was modified to improve the resolving power and reproducibility of separation. The major improvements include stabilization of the electrical current through the device using a voltage divider and stepwise elution of peptide zones in conjunction with the repeated refocusing of remaining peptides. Also, a novel algorithm was developed to calculate more accurately the pI values of peptides identified from experimental data. The standard deviation of calculated pI values for unmodified peptides from the theoretically predicted pI values was on average 0.21 pH units, which is more accurate than in standard-resolution gel-based methods. In order to characterize the analytical performance of the improved device, it was applied for the pI fractionation of yeast proteome digest into 18 fractions, with the collected fractions being analyzed by reverse-phase liquid chromatography coupled with tandem mass spectrometry. Approximately 37% of 20047 identified peptides were detected in only one fraction and 27% - in two fractions. On average, every peptide was found in 2.4 fractions. These results strongly indicate the suitability of the improved device as a first dimension of separation in multidimensional shotgun proteomics analysis, with a potential for fully automated workflow.

First results are reported on the fragmentation of multiply protonated polypeptide ions produced in electrospray ionization mass spectrometry (ESI-MS) with a beam of high-energy cations as a source of activation. The ion beam is generated with a microwave plasma gun installed on a benchtop Q Exactive mass spectrometer. Precursor polypeptide ions are activated when trapped inside the collision cell of the instrument (HCD cell), and product species are detected in the Orbitrap analyzer. Upon exposure to the beam of air plasma cations (∼100 μA, 5 s), model precursor species such as multiply protonated angiotensin I and ubiquitin dissociated across a variety of pathways. Those pathways include the cleavages of C-CO, C-N as well as N-Cα backbone bonds, accordingly manifested as b/y, a, and c/z fragment ion series in tandem mass spectra. The fragmentation pattern observed includes characteristic fragments of collision-induced dissociation (CID) (b/y/a fragments) as well as electron capture/transfer dissociation (ECD, ETD) (c/z fragments), suggesting substantial contribution of both vibrational and electronic excitation in our experiments. Besides backbone cleavages, notable amounts of nondissociated precursor species were observed with reduced net charge, formed via electron or proton transfer between the colliding partners. Peaks corresponding to increased charge states of the precursor ions were also detected, which is the major distinctive feature of ion beam activation.

Recent findings suggest that long-term exposure to diethyl phthalate (DEP), one of the widely used phthalate esters, can lead to serious health problems. Most perfumes contain non-negligible amounts of DEP. Rapid and sensitive detection of DEP in perfumes is thus of increasing importance. A novel procedure based on extractive electrospray ionization mass spectrometry (EESI-MS) has been developed for fast detection and identification of DEP in perfumes without the need for any sample pretreatment. The limit of determination for DEP in perfume was less than 100 ppb using tandem mass spectrometry on a commercial quadrupole time-of-flight mass spectrometer. The dynamic range of this method was about 4 orders of magnitude. A single sample analysis was completed within a few seconds, providing a rapid way to obtain semiquantitative information on the DEP content in perfumes. This study shows that both volatile and nonvolatile analytes (e.g., amino acids) in liquids can be directly sampled by neutral desorption, providing a convenient way for high-throughput screening of target compounds using EESI-MS.