Nanoelectrospray Ionization Source Research Articles

Robust and versatile assembly for emitter positioning, observation, and heating in atmospheric pressure field desorption mass spectrometry.

Atmospheric pressure field desorption (APFD) mass spectrometry (MS) has recently been introduced as a new variant of field desorption (FD) mass spectrometry. The development aimed at providing the basic characteristics of FD-MS in combination with instruments equipped with an atmospheric pressure (AP) interface. Hitherto, APFD has been demonstrated to yield both positive and negative even electron ions of highly polar or ionic compounds as well as to enable the generation of positive molecular ions, M+•, of polycyclic aromatic compounds. The prototype setup for APFD was based on a nano-electrospray ionization (nanoESI) source slightly modified to allow for emitter positioning in front of the AP interface of a Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer. The entrance electrode of the interface was set to negative or positive high voltage with respect to the emitter at ground potential, thereby permitting the formation of positive or negative ions, respectively. This work describes a custom-built device for quicker and more reproducible sample loading on and positioning of field emitters at the entrance electrode of the atmospheric pressure interface of a mass spectrometer. In addition, the device provides means for observation of the emitter during operation and for resistive emitter heating as employed in traditional FD-MS. Emitter heating both speeds up the desorption of the analytes and allows for the desorption/ionization of analytes of higher molecular weight than without emitter heating. In some cases, the signal-to-noise ratio of APFD mass spectra is improved due to higher ion currents effected by compressing the entire process into shorter periods of spectral acquisition. The new setup enables robust and reliable operation in APFD-MS. Moreover, it has been designed as to allow for use on a range of instruments as it can either be used on an FT-ICR mass spectrometer or in combination with a trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) instrument.

Multiplexing Quadrupole and Ion Trap Operation Modes on a "Brick" Miniature Mass Spectrometer.

Although a quadruple mass analyzer and an ion trap mass analyzer have complementary analytical features, they usually have different geometries, operational modes, and electronic control systems. As a continuous effort to extend its coverage, both quadrupole and ion trap operation modes were realized on a "brick" miniature mass spectrometer with a single mass analyzer. In the quadrupole operation mode, low-mass ions ranging from 31 to 502 Th can be analyzed. On the other hand, the ion trap mode can be utilized to cover ions with higher mass to charge ratios (up to 922 Th), as well as performing tandem mass spectrometry. To realize the multiplexing of both operation modes, a printed circuit board (PCB)-based multi-electrode quadrupole-ion trap mass analyzer was designed and integrated in the system. To cover both volatile and non-volatile molecules, two ionization sources were also implemented, including a nano electrospray ionization source and an in-vacuum plasma ionization source. Performances of the instrument operated in these two modes were characterized, such as mass resolution, sensitivity, and mass range. Results demonstrate that the combination of the quadrupole and ion trap operation modes can provide new capabilities when solving analytical problems.

Differential lipid analysis of oxaliplatin-sensitive and resistant HCT116 cells reveals different levels of drug-induced lipid droplet formation.

Lipid droplets (LDs) are intracellular storage vesicles composed of a neutral lipid core surrounded by a glycerophospholipid membrane. LD accumulation is associated with different stages of cancer progression and stress responses resulting from chemotherapy. In previous work, a novel dual nano-electrospray ionization source and data-dependent acquisition method for measuring the relative abundances of lipid species between two extracts were described and validated. Here, this same source and method were used to determine if oxaliplatin-sensitive and resistant cells undergo similar lipid profile changes, with the goal of identifying potential signatures that could predict the effectiveness of an oxaliplatin-containing treatment. Oxaliplatin is commonly used in the treatment of colorectal cancer. When compared to a no-drug control, oxaliplatin dosing caused significant increases in triglyceride (TG) and cholesterol ester (CE) species. These increases were more pronounced in the oxaliplatin-sensitive cells than in oxaliplatin-resistant cells. The increased neutral lipid abundance correlated with LD formation, as confirmed by confocal micrographs of Nile Red-stained cells. Untargeted proteomic analyses also support LD formation after oxaliplatin treatment, with an increased abundance of LD-associated proteins in both the sensitive and resistant cells.

Application of atmospheric pressure field desorption for the analysis of anionic surfactants in commercial detergents

Recent work has shown that field desorption (FD) and field ionization (FI) using activated field emitters may be performed at atmospheric pressure, too. While some limitations apply to atmospheric pressure field desorption (APFD) mass spectrometry (MS), the method can deliver both positive and negative even electron ions of highly polar or ionic compounds. Furthermore, APFD even permits the generation of positive molecular ions of polycyclic aromatic compounds. Here, an application of negative-ion APFD for the analysis of anionic surfactants contained in commercial detergent products for body care, household, and technical uses is presented. The samples include liquid soaps and shower gels, dishwashing liquids, and cooling lubricants. Surfactant solutions in methanol/water or pure methanol at 2–10 µl ml−1 were deposited on commercial 13-µm activated tungsten emitters. The emitters were positioned in front of the atmospheric pressure interface of a Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer by means of a slightly modified nano-electrospray ionization (nanoESI) source. The entrance electrode of the interface was set to positive high voltage with respect to the emitter at ground potential. Under these conditions, negative-ion desorption was achieved. The surfactant anions, organic sulfates and organic sulfonates, were characterized by accurate mass-based formula assignments, and in part, by tandem mass spectrometry. The negative-ion APFD spectra were compared to results by negative-ion electrospray ionization (ESI) either obtained using the FT-ICR mass spectrometer or by using a trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) instrument when product ions of low m/z needed to be detected in tandem MS.Graphical

DiffN Selection of Tandem Mass Spectrometry Precursors.

Current data-dependent acquisition (DDA) approaches select precursor ions for tandem mass spectrometry (MS/MS) characterization based on their absolute intensity, known as a TopN approach. Low-abundance species may not be identified as biomarkers in a TopN approach. Herein, a new DDA approach is proposed, DiffN, which uses the relative differential intensity of ions between two samples to selectively target species undergoing the largest fold changes for MS/MS. Using a dual nano-electrospray (nESI) ionization source which allows samples contained in separate capillaries to be analyzed in parallel, the DiffN approach was developed and validated with well-defined lipid extracts. A dual nESI source and DiffN DDA approach was applied to quantify the differences in lipid abundance between two colorectal cancer cell lines. The SW480 and SW620 lines represent a matched pair from the same patient: the SW480 cells from a primary tumor and the SW620 cells from a metastatic lesion. A comparison of TopN and DiffN DDA approaches on these cancer cell samples highlights the ability of DiffN to increase the likelihood of biomarker discovery and the decreased probability of TopN to efficiently select lipid species that undergo large fold changes. The ability of the DiffN approach to efficiently select precursor ions of interest makes it a strong candidate for lipidomic analyses. This DiffN DDA approach may also apply to other molecule classes (e.g., other metabolites or proteins) that are amenable to shotgun analyses.

Synthesis of large lipid membranes with integrated membrane proteins from gas phase.

In this experimental study I am presenting a new method to generate large, transportable membranes with integrated membrane proteins. These membranes can be used as an in-vitro system to study membrane protein function, investigate its interaction with lipids or study protein complex assembly in a system whose composition is controlled on a molecular basis. The method lends itself to produce anisotropic membranes in the lipid composition and protein orientation. Bio-membranes are generated by self-assembly. There are two established methods - self-assembly on a solid support and self-assembly within a lipo-protein ring: nanodiscs. Both methods solve the principal problem of bio-membrane assembly, their lack of stability in a hydrophilic environment, in a different way. However, solid support based membranes can not be moved from their support and nanodiscs can not be made larger than 17 nm in diameter. Here, large bio-membranes self assemble in a thin sheet of glycerol generated on a liquid surface. I use a nano-electrospray ion source to generate molecular beams of large molecules. The target area is the liquid surface of a container filled with buffer solution. The procedure consists of several steps: 1. formation of a lipid bi-layer; 2. formation of a thin glycerol layer; 3. formation of lipid mono-layer as template for the membrane assembly; 4. adding detergent solubilised membrane proteins and some more lipid; 5. sealing the layers off by some glycerol; 6. detergent extraction from the surface by SM-2 Biobeads over several days. This procedure leads to the formation of a large bio-membrane covering the entire liquid surface which can be transported for inspection or being used for the biochemical activity of the proteins.

A Subtle Change in Nanoflow Rate Alters the Ionization Response As Revealed by Scanning Voltage ESI-MS.

The small charged droplet generated from the nanoelectrospray ionization (nanoESI) source at nL/min flow rate gives its unique feature of high-performance ionization. A continuous scan of the flow rate in this regime can trace the effect of droplet size in greater detail for a better understanding of the ionization process. To date, such practical implementation is hindered by the lack of a suitable liquid pump and the reproducibility of microcapillaries-based systems. Here, offline nanoESI mass spectrometry with a continuously varying flow rate in a dynamic range of several hundred pL/min to ∼100 nL/min was performed by the precision scanning of ESI high voltage (HV). The principle is based on the new paradigm of generating nanoelectrospray from a large Taylor cone with a known spray current-flow rate relationship. The instantaneous flow rate controlled by the HV was determined by simultaneous measurement of the spray current. The system is successfully applied to reveal the role of nanoflow rate on the average charge state of proteins, analysis of analyte mixture, and desalting effect. With the use of a buffer solution with high electric conductivity, a highly controllable oxidative modification was also observed by tuning the flow rate below a threshold of ∼5 nL/min, a finding that has potential application to on-demand oxygen labeling.

Gentle nano-electrospray ion source for reliable and efficient generation of microsolvated ions

We present herein the design of a nano-electrospray ion source capable of reliable generation of large quantities of microsolvated ions. The source is based on a triple molecular skimmer scheme and can be quickly tuned to generate bare ions or their ionic complexes with up to more than 100 solvent molecules retained from solution. The performance of this source is illustrated by recording the mass spectra of distributions of ionic complexes of protonated water, amino acids, and a small protein ubiquitin. Protonated water complexes with more than 110 molecules and amino acids with more than 45 water molecules could be generated. Although the commercial ion source based on the double ion funnel design with orthogonal injection, which we used in our laboratory, is more efficient in generating ions than our triple skimmer ion source, they both exhibit comparable short-term stability in generating bare ions. In return, only the new source is capable of generating microsolvated ions.

Detection and Verification of a Key Intermediate in an Enantioselective Peptide Catalyzed Acylation Reaction.

Until now, the intermediate responsible for the acyl transfer of a highly enantioselective tetrapeptide organocatalyst for the kinetic resolution of trans-cycloalkane-1,2-diols has never been directly observed. It was proposed computationally that a π-methylhistidine moiety is acylated as an intermediate step in the catalytic cycle. In this study we set out to investigate whether we can detect and characterize this key intermediate using NMR-spectroscopy and mass spectrometry. Different mass spectrometric experiments using a nano-ElectroSpray Ionization (ESI) source and tandem MS-techniques allowed the identification of tetrapeptide acylium ions using different acylation reagents. The complexes of trans-cyclohexane-1,2-diols with the tetrapeptide were also detected. Additionally, we were able to detect acylated tetrapeptides in solution using NMR-spectroscopy and monitor the acetylation reaction of a trans-cyclohexane-1,2-diol. These findings are important steps towards the understanding of this highly enantioselective organocatalyst.

Development of an In-Source Peltier Heater for Pulled Capillary Nanospray Emitter Columns.

Column compartments in liquid chromatography (LC) systems house the LC columns. These compartments are responsible for maintaining a suitable column environment for achieving optimal chromatographic performance. However, the advancements in instrument and column designs demand newer technologies. It is a well-established concept that decreasing the dead volume of the column improves the column resolution, thereby providing enhanced chromatographic separation. One of the major contributors in the dead volume is the line connecting the column in the LC compartment to the ion source in the mass spectrometer. Using in-source emitter columns is one strategy to enhance the resolution. However, ion sources without temperature control are not suitable for columns that are used at high temperatures. In this work, we are introducing a nano electrospray ionization source with an integrated Peltier heater designed for pulled capillary nanospray emitter columns. Although the performance of the device is demonstrated by showing the isomeric separation of permethylated glycans using a mesoporous graphitized carbon packed pulled capillary emitter, it can easily be paired with any nanospray emitter column that requires temperature control.

Updated clinical and glycomic features of mannosyl-oligosaccharide glucosidase deficiency: Two case reports

BACKGROUNDMannosyl-oligosaccharide glucosidase (MOGS) deficiency is an extremely rare type of congenital disorder of glycosylation (CDG), with only 12 reported cases. Its clinical, genetic, and glycomic features are still expanding. Our aim is to update the novel clinical and glycosylation features of 2 previously reported patients with MOGS-CDG.CASE SUMMARYWe collected comprehensive clinical information, and conducted the immunoglobulin G1 glycosylation assay using nano-electrospray ionization source quadruple time-of-flight mass spectrometry. Novel dysmorphic features included an enlarged tongue, forwardly rotated earlobes, a birth mark, overlapped toes, and abnormal fat distribution. Novel imaging findings included pericardial effusion, a deep interarytenoid groove, mild congenital subglottic stenosis, and laryngomalacia. Novel laboratory findings included peripheral leukocytosis with neutrophil predominance, elevated C-reactive protein and creatine kinase, dyslipidemia, coagulopathy, complement 3 and complement 4 deficiencies, decreased proportions of T lymphocytes and natural killer cells, and increased serum interleukin 6. Glycosylation studies showed a significant increase of hypermannosylated glycopeptides (Glc3Man7GlcNAc2/N2H10 and Man5GlcNAc2/N2H5) and hypersialylated glycopeptides. A compensatory glycosylation pathway leading to an increase in Man5GlcNAc2/N2H5 was indicated with the glycosylation profile. CONCLUSIONWe confirmed abnormal glycomics in 1 patient, expanding the clinical and glycomic spectrum of MOGS-CDG. We also postulated a compensatory glycosylation pathway, leading to a possible serum biomarker for future diagnosis.

Zinc-binding proteins in stallion seminal plasma as potential sperm function regulators

Abstract Sperm functions may be influenced by seminal plasma (SP) proteins with affinity to zinc ions. The aim of the study was to isolate and characterise zinc-binding proteins (ZnBPs) from stallion SP using proteomic tools. Zinc-binding proteins were isolated from the SP of eight stallions by zinc-affinity chromatography. They were analysed in the SDS-PAGE system, and peptide extracts were prepared. Samples of ZnBPs isolated from stallion SP were injected onto the nLC-1000 nanoflow HPLC system coupled via a nano-electrospray ion source to the Orbitrap Elite FTMS mass spectrometer (Thermo Fisher Scientific). Raw MS data were analysed using MaxQuant software with label-free quantification (LFQ). Protein sequences were obtained from the UniProt database. Forty-seven proteins in ZnBPs were annotated in a gene ontology (GO) analysis. The LFQ intensity analysis of individual proteins revealed that ZnBPs comprised mainly clusterin (CLU, 27%), serin protease inhibitor F1 (SERPINF1, 13%), actin, cytoplasmic 1 (ACTB, 9%), nucleobindin 2 (NUCB2, 8%) and polymeric immunoglobulin receptor (PIGR, 6%). This is the first proteomic study to analyse ZnBPs in stallion SP. The present findings show that ZnBPs in stallion SP could play an important role in the regulation of sperm function.

Portable nanoelectrospray periodic focusing differential ion mobility spectrometer for the rapid screening of illicit drugs

The spread of illicit drug abuse is a major public health issue in most countries. Law enforcement personnel on drug abuse need a tool to confiscate and block illicit trade of illicit drug or monitoring drug impaired driving. Therefore, affordable and portable instruments for illegal drug detection are in critical need. In this study, we developed a portable periodic focusing differential ion mobility (PFDMA) spectrometer for in situ motoring illicit drugs. The device comprised an ionization source, drift cell, air blower, voltage divider board and electronic control board. In general, an ion mobility spectrometer (IMS) is able to work with air as a buffer gas at atmospheric pressure. There is no need to install heavy mechanical and/or expensive turbo molecular pumps. For our instrument, a small air blower was used to generate laminar counterflow. The whole device is compact with a size of 150 × 200 × 280 (WLH, mm) and a weight of 2.5 kg. In general, most commercial drift tube ion mobility spectrometers use radioactive or corona discharge ionization. In this work, we utilized a different approach to analyze liquid samples. The device was equipped with a nanoelectrospray ionization source. To avoid memory effects, a disposable micropipette was used. An aluminum-coated tip with an ID of 2 μm was applied with a high voltage to achieve nanoelectrospray ionization without sheath gas. Advantages of PFDMA are to isomer separation and better sensitivity compared to other types of ion mobility spectrometers. Most illicit drugs could be detected by this homemade device. The detection limit is lower than the level allowed by law. With our device, the detection limit of methamphetamine is ∼10 ng/ml.

Automated Multidimensional Nanoscale Chromatography for Ultrasensitive Targeted Mass Spectrometry.

Recent advances in nanoscale separations and high-resolution mass spectrometry permit highly sensitive and accurate analyses of complex protein mixtures. Here, we describe improved methods for nanoscale multidimensional chromatography coupled to targeted mass spectrometry (tMS) to achieve ultrasensitive quantification of peptides in complex proteomes. The presented chromatographic system consists of capillary strong-cation exchange (SCX) chromatography column, from which peptides are eluted directly onto high-resolution reversed-phase (RP) analytical columns and nanoelectrospray ion source. SCX prefractionation is used to separate phosphorylated peptides, permitting their ultrasensitive quantification. Resolution and robustness of this chromatographic system, together with the orthogonality of SCX and RP separations, permit scheduling of large panels of targeted MS assays. This design also enables seamless scaling to three-dimensional separations, thereby enabling large-scale, ultrasensitive quantitative proteomics.

Protein Profiling Identify Distinct Tumor-Tissue Protein Expression Pattern Specific for Relapsing Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease. Several genetic drivers have been suggested based on gene-expression and RNA sequencing. However, clinical trials based on these molecular subclassifications have failed to improve treatment results and standard of care in DLBCL is still R-CHOP (rituximab, cyclophosphamide, doxorubicin, prednisone) based therapy, which results in approximately an 70% 5-year overall survival rate. Nordic Lymphoma Group (NLG) has conducted two large phase II trials (NLG-LBC-04; NCT 01502982 and NLG-LBC-05; NCT 01325194) during the last decade with the aim of improving the treatment outcome for young (<65 years) high-risk patients with primary DLBCL. Both protocols were based on biweekly administered R-CHOP-14 with etoposide (R-CHOEP) and systemic central nervous system (CNS) prophylaxis. Various factors acting at different biological levels may influence pathology of the disease, and discrepancies between gene expression and the final combination of the functional proteins may be present due to, e.g., translational inhibition/activation and posttranslational modifications. Mass spectrometry-based protein profiling is a high throughput methodology that enables identification of the proteins in a given cell or tissue and the global protein expression can be compared between patients.The aim of our study was to search for prognostic markers and possible therapeutic targets at the protein level in a uniformly treated patient cohort from the two Nordic trials (n=64) with available tumor-tissue. Thus, in this study, we investigated the protein expression pattern in the pre-therapeutic formalin-fixed paraffin embedded tumor samples by nano liquid-chromatography coupled to a mass spectrometer (Orbitrap Fusion) through an EASY-Spray nano-electrospray ion source (nLC-MS/MS). We identified 4,622 proteins with at least two unique peptides across all samples. In the combined cohort, we were able to, based on differential protein expression of 190 proteins between patients (p<0.05) with treatment sensitive (n=53) and relapsing (n=11) lymphoma, identify three clusters with one of the clusters containing only patients with experienced relapsing disease. Interestingly, among other identified differentially expressed proteins, we emphasize the identification of voltage dependent anion channel 3 (VDAC3) and the GTPase RAC1. This pattern of protein expression suggests disturbance in reactive oxidative species (ROS) signaling and regulation of tumor cell proliferation between relapsing and therapy-responding patients. Assessment of these specific protein expression patterns in the three clusters may provide a useful parameter for prediction of relapsing disease in DLBCL patients but still awaits further validation in a separate patient cohort. DisclosuresHolte: Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Nordic: Membership on an entity's Board of Directors or advisory committees; Nanovector: Membership on an entity's Board of Directors or advisory committees, Other: lectures honorarias; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Jørgensen: Gilead/Kite: Consultancy; Novartis: Consultancy; Celgene/BMS: Consultancy; Roche: Consultancy.

Novel Insight into Proximal DNA Domain Interactions from Temperature‐Controlled Electrospray Ionization Mass Spectrometry

AbstractQuadruplexes are non‐canonical nucleic acid structures essential for many cellular processes. Hybrid quadruplex–duplex oligonucleotide assemblies comprised of multiple domains are challenging to study with conventional biophysical methods due to their structural complexity. Here, we introduce a novel method based on native mass spectrometry (MS) coupled with a custom‐built temperature‐controlled nanoelectrospray ionization (TCnESI) source designed to investigate interactions between proximal DNA domains. Thermal denaturation experiments were aimed to study unfolding of multi‐stranded oligonucleotide constructs derived from biologically relevant structures and to identify unfolding intermediates. Using the TCnESI MS, we observed changes in Tm and thermodynamic characteristics of proximal DNA domains depending on the number of domains, their position, and order in a single experiment.

Novel Insight into Proximal DNA Domain Interactions from Temperature-Controlled Electrospray Ionization Mass Spectrometry.

Quadruplexes are non‐canonical nucleic acid structures essential for many cellular processes. Hybrid quadruplex–duplex oligonucleotide assemblies comprised of multiple domains are challenging to study with conventional biophysical methods due to their structural complexity. Here, we introduce a novel method based on native mass spectrometry (MS) coupled with a custom‐built temperature‐controlled nanoelectrospray ionization (TCnESI) source designed to investigate interactions between proximal DNA domains. Thermal denaturation experiments were aimed to study unfolding of multi‐stranded oligonucleotide constructs derived from biologically relevant structures and to identify unfolding intermediates. Using the TCnESI MS, we observed changes in T m and thermodynamic characteristics of proximal DNA domains depending on the number of domains, their position, and order in a single experiment.

Qualitative screening of prohibited drugs in dietary supplements using a homemade miniature mass spectrometer

Qualitative screening of prohibited drugs in dietary supplements based on a miniature homemade mass spectrometer was presented, and its application for real health sample analyses was demonstrated in this work. To alleviate the labor and equipment-intensive procedures of traditional detection methods, simplified sample preparation procedures were coupled with one-time use Nano electrospray ionization sources for practical sample analyses. A small tandem mass spectrometry database was built for 16 illegal adulterants, so that these prohibited drugs could be quickly screened in different samples. Tandem mass spectrometry results were compared with a commercial orbitrap mass spectrometer. After exploring some possible factors, such as the sonication duration, the reproducibility of the method and the effect of matrix on sensitivity, the instrument and dataset were then used for the screening of these 16 adulterants in five commercial products: one weight loss product and four products claiming to strengthen immunity. Illicit adulterants were detected in the weight loss product but not in other samples. These results were also confirmed using the conventional liquid chromatography-mass spectrometry technique.

3D-Printed Microfluidic Nanoelectrospray Ionization Source Based on Hydrodynamic Focusing.

Nanoelectrospray ionization (nESI) mass spectrometry (MS) is an ideal detection method for microfluidic chips, and its performances depend on nESI emitters. However, the fabrication of monolithic nESI emitters in chips was difficult. Herein, we propose a three-dimensional (3D) printing method to develop a microfluidic nanoelectrospray ionization source (NIS), composed of a nESI emitter and other components. Firstly, the NIS was compatible with a 50 - 500 nL min-1 nanoflows by imposing 3D hydrodynamic focusing to compensate for the total flow rate, achieving a 7.2% best relative standard deviation in the total ion current (TIC) profiles. Additionally, it was applied to probe thirteen organic chemicals, insulin, and lysozyme with adequate signal-to-noise ratios and an accuracy of m/z between 9.02 × 10-1 and 1.48 × 103 ppm. Finally, the NIS achieved comparable limits of detection compared with its commercial counterpart. Considering the standardized preparation of NIS, it would be a potential option to develop 3D-printed customized Chip-MS platforms.

A Gas-Phase Reaction Accelerator Using Vortex Flows.

Gas-phase microdroplets have been increasingly used for reaction acceleration. Here, we report the development of a vortex tube as a reaction accelerator. Three types of reactions, viz., aromatization, amination isomerization, and acid-induced cytochrome c unfolding were used to characterize the performance of the vortex tube. During ion transfer from a nanoelectrospray ionization (nanoESI) source to the mass spectrometry (MS) inlet, the generated vortex flows helped droplet desolvation and ion confinement and thus improved the MS intensity by 2-3 orders of magnitude compared with that when the vortex tube was not applied. Like the stirring effect in the bulk phase, the reactants were more sufficiently mixed and reacted in vortices. Therefore, with the same reaction distance, a 2-3-fold improvement of conversion ratios was observed by using the vortices. Notably, the vortex tube enabled the use of flow rate to control the reaction time for ∼60 μs, which was useful for precise control of reaction progress. As a demonstration, the intermediates of the amination isomerization were tracked and the equilibrium constant and rate constant of the cytochrome c unfolding were determined.