Corrigendum to "Advancing mass spectrometry-based chemical imaging: A noncontact continuous flow surface probe in mass spectrometry for enhanced signal detection and spatial resolution" [Talanta 273 (2024) 125858].

In the statement "This innovative MS imaging system can be directly applied to real tissue systems and other plant samples to visualize the molecular level distributions." "innovative" should be read as "important".

Advancing mass spectrometry-based chemical imaging: A noncontact continuous flow surface probe in mass spectrometry for enhanced signal detection and spatial resolution.

A new method has been developed for mass spectrometric imaging of small molecules and proteins on tissue or in thinly sliced materials. A laser desorption Venturi electrospray ionization-mass spectrometer was developed for molecular imaging. This method combines laser desorption (LD) and electrospray ionization (ESI) systems before a mass spectrometer (MS). To carry out laser desorption, samples are excited with a laser from the back side of a glass substrate. The desorbed molecules or particles are then captured by a solvent flow. In the ESI system, these desorbed particles and molecules are ionized. The spray part of the solvent system consists of two capillaries: one delivers solvent to the sample plate sides to capture desorbed molecules and particles, and the other carries the solution to the mass spectrometry side using the Venturi effect. A 2D stage facilitates sampling. The system is designed to minimize the sample size after desorption using a 355 nm diode laser, and it is optimized for molecules of various sizes, including organic molecules, amino acids, and proteins. Despite challenging atmospheric conditions for protein desorption, this specialized design enables the collection of protein spectra. The amino acids and other small molecules showed high sensitivity in the MSI measurements. This innovative MS imaging system can be directly applied to real tissue systems and other plant samples to visualize the molecular level distributions.

High-performance peptide and disulfide mapping by direct injection of intact proteins using on-line coupled UV-liquid chromatography microdroplet mass spectrometry (UVLC-MMS).

Microdroplet mass spectrometry (MMS), achieving ultra-fast enzyme digestion in the ionization source, holds great promises for innovating protein analysis. Here, in-depth protein characterization is demonstrated by direct injection of intact protein mixtures via on-line coupling MMS with capillary C4 liquid chromatography (LC) containing UV windows (UVLC-MMS) through an enzyme introduction tee. We showed complete sets of peptides of individual proteins (hemoglobin, bovine serum albumin, and ribonuclease A) in a mixture could be obtained in one injection. Such full (100%) sequence coverage, however, could not be achieved by conventional nanoLC-MS method using bottom-up approach with single enzyme. Moreover, direct injection of a chaperone α-crystalline (α-Cry) complex yielded identification of post-translational modifications including novel sites and semi-quantitative characterization including 3:1 stoichiometry ratio of αA- and αB-Cry sub-units and ∼1.4 phosphorylation/subunit on S45 (novel site) and S122 (main site) of αA-Cry, ∼0.7 phosphorylation/subunit on S19 (main site) and S45 of αB-Cry, as well as 100% acetylation on both N-termini of each subunits by matching the mass and retention time of the intact and its digested peptides. Furthermore, trifluoroacetic acid was able to be used in the mobile phase with UVLC-MMS to improve the separation of differentially reduced intact species and detectability of the droplet-digested products. This allowed us to completely map four disulfide linkages of ribonuclease A based on collision-induced dissociation of disulfide clusters, some of which would otherwise not be detected, preventing scrambling or shuffling errors arising from lengthy bulk solution digestion by the bottom-up approach. Integration of UVLC and MMS greatly improves droplet digestion efficiency and MS detection, enabling highly efficient workflow for in-depth and accurate protein characterization.

Simultaneous detection of positively and negatively charged microparticles by ion trap mass spectrometry.

This study describes the simultaneous detection of positively and negatively charged microparticles by ion trap mass spectrometry (IT-MS) as a novel analytical measurement technique. The instrument was configured with a feeding capillary for particle introduction, an ion trap, and a charge detector that responds to both ions simultaneously. Positively and negatively charged particles are generated by the triboelectric effect inside the capillary entrance of the instrument. The particles were fed in dry form with a cotton tip to provide the best dispersion. No potential was applied to the lenses on the path of particles and end caps on the ion trap. Particle size calibration has been done using well-defined polystyrene spheres in different sizes. For this study, 2 µm standard polystyrene (PS) spheres were used and checked by different particle sizes. A charge detector detected the ejected positive and negative ions, and the results were evaluated by a program that works under the Labview. The positive and negative ions reached the detector sequentially with respect to their m/z amount. The masses of particles were determined depending on their arrival time at the detector. The IT-MS system and charge detector simultaneously allow positively and negatively charged particles to be detected. This is the first study in the literature that simultaneously shows the trapping and detection of oppositely charged particles.

Revisiting Disulfide-Yne and Disulfide-Diazonium Reactions for Potential Direct Modification of Disulfide Bonds in Proteins.

To find their potential use in protein research, direct addition of a disulfide compound to alkyne (namely disulfide-yne reaction) and S-arylation with arenediazonium salt (namely disulfide-diazonium reaction) were investigated in aqueous or protic solutions. The reaction of dimethyl disulfide with 5-hexynol performed best under 300 nm irradiation in the presence of sodium acetate to afford 5,6-bis(methylthio)-5-hexenol in 60% yield. Without the prior reduction of a disulfide bond to thiols, the disulfide-yne reactions have the advantage of 100% atom economy. Disulfide-diazonium reaction was triggered by sodium formate and accelerated by photoirradiation with a 450 nm LED lamp (5 W). The reaction of 3,4-dihydroxy-1,2-dithiane with 2-(prop-2-yn-1-yloxy)benzene-1-diazonium tetrafluoroborate (8b) afforded 2-(benzofuran-3-yl)-1,3-dithiepane-5,6-diol (13), confirming that both S substituents originate from the same disulfide molecule. The trastuzumab antibody was incubated with diazonium 8b, followed by α-lytic protease digestion, LC-ESI-MS/MS analysis, and Mascot search, to verify that the proximal C229 and C232 residues on the same heavy chain were reconnected with a (benzofuranyl)methine moiety that originated from 8b, unlike the expected disulfide rebridging across two heavy chains. Nonetheless, disulfide-diazonium reactions still have potential for rebridging disulfide bonds if appropriate proteins and diazonium agents are chosen.

Harnessing natural-product-inspired combinatorial chemistry and computation-guided synthesis to develop N-glycan modulators as anticancer agents.

Modulation of N-glycosylation using human Golgi α-mannosidase II (α-hGMII) inhibitors is a potential anticancer approach, but the clinical utility of current α-hGMII inhibitors is limited by their co-inhibition of human lysosomal α-mannosidase (α-hLM), resulting in abnormal storage of oligomannoses. We describe the synthesis and screening of a small library of novel bicyclic iminosugar-based scaffolds, prepared via natural product-inspired combinatorial chemistry (NPICC), which resulted in the identification of a primary α-hGMII inhibitor with 13.5-fold selectivity over α-hLM. Derivatization of this primary inhibitor using computation-guided synthesis (CGS) yielded an advanced α-hGMII inhibitor with nanomolar potency and 106-fold selectivity over α-hLM. In vitro studies demonstrated its N-glycan modulation and inhibitory effect on hepatocellular carcinoma (HCC) cells. In vivo studies confirmed its encouraging anti-HCC activity, without evidence of oligomannose accumulation.

Portable particle mass spectrometer.

Particle pollutants in air have been confirmed to damage human health. The PM10 concentration is an important parameter for air quality determination. In this study, a portable quadrupole ion trap mass spectrometer (QIT-MS) was developed and used to quantitate microparticles and particulate standards. The instrument can be used to perform online analysis of various microsized particles. The instrument can be used to analyze various sizes of disperse particles with accurate mass by a histogram profile. The overall detection efficiencies of particles in the sample for polystyrene were obtained. PM10-like reference materials were used for calibration to analyze the size and mass distribution of an environmental sample. The instrument shows the potential for quantitation of different particles of an unknown sample.

Nanodiamond Solid-Phase Extraction and Triton X-114 Cloud Point Separation for Robust Fractionation and Shotgun Proteomics Analysis of the Human Serum Proteome.

Human serum is one of the most attractive specimens in biomarker research. However, its overcomplicated properties have hindered the analysis of low-abundance proteins by conventional mass spectrometry techniques. This work proposes an innovative strategy for utilizing nanodiamonds (NDs) in combination with Triton X-114 protein extraction to fractionate the crude serum to six pH-tuned fractions, simplifying the overall proteome and facilitating protein profiling with high efficiency. A total of 663 proteins are identified and evenly distributed among the fractions along with 39 FDA-approved biomarkers─a remarkable increase from the 230 proteins found in unfractionated crude serum. In the low-abundance protein section, 88 proteins with 7 FDA-approved biomarkers are detected─a marked increase from the 15 proteins (2 biomarkers) observed in the untreated sample. Notably, fractions at pH 11, derived from the aqueous phase of detergent separation, suggest potential applications in rapid and robust serum proteome analysis. Notably, by outlining the excellent properties of NDs for proteomic research, this work suggests a promising extraction protocol utilizing the great compatibility of NDs with streamlined serum proteomics and identifies potential avenues for future developments. Finally, we believe that this work not just improves shotgun proteomics but also opens up studies on the interaction between NDs and the human proteome. Data are available via ProteomeXchange with the identifier PXD029710.

Investigation of solvent microparticle formation in spray ionization-quadrupole ion trap-mass spectrometry.

In the present study, a new method has been developed for the real-time analysis of insource created solvent particles based on spray ionization-quadrupole ion trap-mass spectrometry (SI-QIT-MS). This is the first work in the literature reporting the formation of different solvent particles during solvent spray in mass spectrometry. The solvent particles formed from the solvent droplets are detected by a charge detector. Our ion trap system allows the measurement of a wide range particle masses. Various solvents and solvent mixtures such as water, methanol, acetone, toluene, n-butanol, water-methanol, and water-ethanol were sprayed through a cone system, and the mass of the particles was monitored by different trap frequencies and voltages. While polar molecules produce larger and more diverse particles due to their strong intermolecular forces, apolar solvents generally do not produce a significant number of particles. We obtained results using a homemade ion trap mass spectrometer capable of determining the mass of micro-sized solvent and solvent mixture particles weighing up to 1015 (Da). The instrument uses a charge detector connected to the exit of the ion trap. Simultaneous acquisition of particle mass spectra and measurement of the amount of charge in each particle allow mass assignment of each particle. Sprayed solvent particles were examined at various trap frequencies and voltages to find the best instrumental parameters for the highest trapping efficiency. The custom SI-QIT-MS instrument allows the measurement of the mass distribution of charged particles from the solvent spray.

Development of a focused high-energy macromolecular ion beam.

In this work, we report the development of a focused macromolecular ion beam with kinetic energy of up to 110 keV. The system consists of a quadrupole ion trap (QIT), einzel lens and linear accelerator (LINAC). Based on the combination of matrix-assisted laser desorption ionization (MALDI) and quadrupole ion trapping (QIT), ions were desorbed from the surface and trapped with an ion trap to form biomolecular ion packets. Positive- and negative-pulsed voltages were applied on each end-cap electrode of the QIT to extract the ion packets and form an ion beam that was subsequently focused via an einzel lens and accelerated by stepwise pulsed voltages. The tabletop instrument was designed and successfully demonstrated via measurements of molecular ions of insulin, cytochrome c and bovine serum albumin (BSA) with mass-to-charge ratios (m/z) ranging from ∼5.8 to 66.5 k. This is the first report of both a focused and high-kinetic-energy protein ion beam. In addition, both secondary ions and electrons were observed from the surface by hypervelocity ion beam bombardment. This focused macromolecular ion beam has demonstrated its potential in the study of interactions between large molecular ions with other molecules either in the gas phase or upon a surface.

Comprehensive Workflow for Mapping Disulfide Linkages Including Free Thiols and Error Checking by On-Line UV-Induced Precolumn Reduction and Spiked Control.

Mapping highly complicated disulfide linkages and free thiols via liquid chromatography-tandem mass spectrometry (LC-MS2) is challenging because of the difficulties in optimizing sample preparation to acquire critical MS data and detecting mispairings. Herein, we report a highly efficient and comprehensive workflow using an on-line UV-induced precolumn reduction tandem mass spectrometry (UV-LC-MS2) coupled with two-stage data analysis and spiked control. UV-LC-MS2 features a gradient run of acetonitrile containing a tunable percentage of photoinitiators (acetone/alcohol) that drives the sample to the MS through a UV-flow cell and reverse phase column to separate UV-induced products for subsequent fragmentation via low energy collision-induced dissociation. This allowed the alkylated thiol-containing and UV-reduced cysteine-containing peptides to be identified by a nontargeted database search. Expected or unexpected disulfide/thiol mapping was then carried out based on the search results, and data were derived from partially reduced species by photochemical reaction. Complete assignments of native and scrambled disulfide linkages of insulin, α-lactalbumin, and bovine serum albumin (BSA) as well as the free C34-BSA were demonstrated using none or single enzyme digestion. This workflow was applied to characterize unknown disulfide/thiol patterns of the recombinant cyclophilin 1 monomer (rTvCyP1 mono) from the human pathogen Trichomonas vaginalis. α-Lactalbumin was judiciously chosen as a spiked control to minimize mispairings due to sample preparation. rTvCyP1 was determined to contain a high percentage of thiol (>80%). The rest of rTvCyP1 mono were identified to contain two disulfide/thiol patterns, of which C41-C169 linkage was confirmed to exist as C53-C181 in rTvCyP2, a homologue of rTvCyP1. This platform identifies heterogeneous protein disulfide/thiol patterns in a de-novo fashion with artifact control, opening up an opportunity to characterize crude proteins for many applications.

Enhancement of fucosylated N-glycan isomer separation with an ultrahigh column temperature in porous graphitic carbon liquid chromatography-mass spectrometry.

Due to the heterogeneous and isomeric nature of glycans, the development of an advanced separation of distinct glycan isomers is essential for glycan research and application. In this study, we utilized porous graphite carbon (PGC) chromatography for the separation of isomeric oligosaccharides without reduction or chemical derivatization at 190 °C in a custom-built heating oven. Furthermore, the fine structures of glycan isomers could be identified by using ultrahigh temperature PGC liquid chromatography mass spectrometry (UHT-PGC-LCMS). A nonreduced hydrolyzed dextran was applied to verify the performance of UHT-PGC. When the temperature of the PGC column was increased from 25 to 190 °C, the liquid chromatography separation power of the nonreduced dextran ladder significantly increased. The advantage of the UHT-PGC column was its high peak capacity with gradient elution in 10 min at 190 °C, 6700 psi, and a 250 µL/min flow rate for native glycan analysis. Four synthetic Lewis antigen isomers were used to elucidate the separation effectiveness in UHT-PGC. Moreover, mass spectrometry-based sequencing to generate specific diagnostic ions from the four synthetic Lewis antigens was used to predict isomeric glycans based on the relative intensity ratio (RIR) of diagnostic ions. The intensities of the diagnostic ions of synthetic isomers were used to identify each isomer of the fucosylated glycan. The results clearly showed that terminal Lewis A and X residues were in the 3- and 6-arms of N-glycan, respectively.

A portable multiple ionization source biological mass spectrometer.

In the past, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), used for large biomolecule detection, were usually installed in two separate mass spectrometers. In this study, they were equipped in the same mass spectrometer. This portable biological mass spectrometer has multiple ionization capabilities in the same mass spectrometer and shares the same mass analyzer and detector. This mass spectrometer can be operated under low vacuum (∼10-3 Torr) and can use air as the buffer gas. Therefore, the demand for pumping is reduced and rare gas feeding is no longer essential. A small scroll pump, employed to assist a miniature turbo pump, is sufficient to maintain the operational pressure. The mass spectra of biomolecules were obtained using frequency scanning instead of voltage ramping. Therefore, a wider mass-to-charge ratio (m/z) range was achieved. Furthermore, the design also couples a conversion dynode with a channeltron to enhance the mass detection range. This homemade mass spectrometer has the capability to measure charged particles with very large m/z values (m/z > 100 000). The concentrations of the studied compounds (angiotensin, insulin, cytochrome C, bovine serum albumin (BSA), immunoglobulin G, and immunoglobulin A) are from 5 femtomole to 100 picomole, and the mass resolutions are from 30 to 260. The mass range of this portable mass spectrometer was comparable with a commercial linear time-of-flight mass spectrometer owing to the use of the frequency scan.

A quadrupole ion trap mass spectrometer for dry microparticle analysis.

In this work, we report a new design of a charge detection quadrupole ion trap mass spectrometer (QIT-MS) for the analysis of micro-sized dry inorganic and bioparticles including red blood cells (RBCs) and different sizes of MCF-7 breast cancer cells. The developed method is one of the fastest methods to measure the mass of micro-sized particles. This system allows the online analysis of various micro-sized particles up to 1 × 1017 Da. The calibration of the mass spectrometer has been done by using different sizes of polystyrene (PS) particles (2-15 µm). The measured masses of RBCs were around 1.8 × 1013 Da and MCF-7 cancer cells were between 1 × 1014 and 4 × 1014 Da. The calculated mass distribution profiles of the particles and cells were given as histogram profiles. The statistical data were summarized after Gaussian type fitting to the experimental histogram profiles. The new method gives very promising results for the analysis of particles and has very broad application.

Biomolecular Clusters Distribution up to Mega Dalton Region Using MALDI-Quadrupole Ion Trap Mass Spectrometer.

We present the first report on complete cluster distributions of cytochrome c (molecular weight of 12.4 kDa) and bovine serum albumin ((BSA), molecular weight of 66.4 kDa) with mass-to-charge ratio (m/z) reaching 350,000 and 1,400,000, respectively, by matrix-assisted laser desorption/ionization (MALDI). Large cluster distributions of the analytes were measured by our homemade frequency-scanned quadrupole ion trap (QIT) mass spectrometer with a charge detector. To our knowledge, we report the highest m/z clusters of these two biomolecules. The quantitative results indicate that large clusters ions of cytochrome c and BSA follow the power law (r² > 0.99) with cluster size distribution, which provides experimental evidence for the laser ablation studies of MALDI.

Investigation of manganese(II)-insulin complexes using electrospray ionization mass spectrometry.

Manganese is a trace element in human nutrition. It is involved in many enzymes, proteins and biological activities. Mn(II) ion has the capable of binding to protein or peptides. Insulin is a blood glucose-lowering peptide hormone and it is secreted by the pancreatic ß-cells. In this study, the binding capability of Mn(II) ions to insulin was studied using ESI-MS, nano-ESI-MS and MS/MS methods. The binding of Mn(II) ions to insulin molecule was studied by examining the effect of pH, the molar ratio of Mn(II) ions to insulin, the flow rate with nano-ESI system and MS/MS spectrometry. The ESI-MS measurements showed that the Mn(II)-insulin complexation mostly produces ML and M2L type complexes. The highest binding ratio was found at pH 7. The complex formation equilibrium constants of Mn(II)-insulin were calculated as Kf1: 1.03 ±â€¯0.12 × 104 and Kf2: 1.93 ±â€¯0.17 × 103. The nano-ESI-MS and MS/MS measurements exhibited strong and specific binding of Mn(II) ions to insulin molecule. It was concluded from all the ESI-MS measurements that Mn(II) ion has a high affinity to insulin molecule to form stable complexes.

ESI-MS measurements for the equilibrium constants of copper(II)-insulin complexes.

Trace elements regulate many biological reactions in the body. Copper(II) is known as one of trace elements and capable of binding to proteins. Insulin is a blood glucose-lowering peptide hormone and it is secreted by the pancreatic ß-cells. In this study, Cu(II)-insulin complexes were investigated by using ESI-MS method. Insulin molecule gives ESI-MS peaks at +4, +5, +6 and +7 charged states. Cu(II)-insulin complexes can be monitored and quantified on the ESI-MS spectra as the shifted peaks according to insulin peaks. The solutions of Cu(II)-insulin complexes at different pHs and mole ratios of Cu(II) ions to insulin molecule were measured on the ESI-MS. The highest complex formation ratio for Cu(II)-insulin were found at pH 7. The multiple bindings of Cu(II) ions to insulin molecule was observed. The formation equilibrium constants of Cu(II)-insulin complexes were calculated as Kf1: 3.34 × 104, Kf2: 2.99 × 104, Kf3: 7.00 × 103 and Kf4:2.86 × 103. The specific binding property of Cu(II) ions was controlled by using different spray ion sources including electrospray and nano-electrospray. The binding property of Cu(II) also investigated by MS/MS fragmentation. It was concluded from the ESI-MS measurements that Cu(II) ion has a high affinity to insulin molecules to form stable complexes.

ESI MS for Microsized Bioparticles.

An ESI ion trap mass spectrometer was designed for high-throughput and rapid mass analysis of large bioparticles. Mass calibration of the instrument was performed using commercially available polystyrene (PS) microparticles with a size comparable to cancer cells. Different sizes of MCF-7 breast cancer cells (8 to 15 µm) were used in this study. The masses of different cancer cells were measured. This system allows for the analysis of all types of particles.

Substrate Preference and Interplay of Fucosyltransferase 8 and N-Acetylglucosaminyltransferases.

The core fucosylation of N-glycans on glycoproteins is catalyzed by fucosyltransferase 8 (FUT8) in mammalian cells and is involved in various biological functions, such as protein function, cancer progression, and postnatal development. The substrate specificity of FUT8 toward bi-antennary N-glycans has been reported, but it is unclear with regard to tri-antennary and tetra-antennary glycans. Here, we examined the specificity and activity of human FUT8 toward tri- and tetra-antennary N-glycans in the forms of glycopeptides. We found that the tri-antennary glycan [A3(2,4,2) type] terminated with N-acetylglucosamine (GlcNAc), which is generated by N-acetylglucosaminyltransferase (GnT)-IV, is a good substrate for FUT8, but the A3(2,2,6) type of tri-antennary glycan, generated by GnT-V, is not a substrate for FUT8. We also observed that core fucosylation reduced the activity of GnT-IV toward the bi-antennary glycan. Examining the correlation between the types of N-glycans and the expression levels of FUT8, GnT-IV, and GnT-V in cells revealed that these glycosyltransferases, particularly GnT-IV, play important roles in directing the branching and core fucosylation of N-glycans in vivo. This study thus provides insights into the interplay among FUT8, GnT-IV, and GnT-V in N-linked glycosylation during the assembly of glycoproteins.

Quantifying Na(I)-insulin and K(I)-insulin non-covalent complexes by ESI-MS method and calculation of their equilibrium constants.

In this study, the dissociation and formation equilibrium constants of Na(I)-insulin and K(I)-insulin complexes have been calculated after the quantifying them on ESI mass spectrometer. The ESI-MS spectra of the complexes were measured by using the solvents as 50% MeOH in water and 100% water. The effect of pH on the Na(I)-insulin and K(I)-insulin complex formation were examined. Serial binding of Na(I) and K(I) ions to the insulin molecule were observed in the ESI-MS measurements. The first formation equilibrium constants were calculated as Kf1: 5.48×103 1/M for Na(I)-insulin complex and Kf1: 4.87×103 1/M for K(I)-insulin in water. The binding capability of Na(I) ions to insulin molecule is higher than the capability of K(I) ions. In case of a comparison together with Ca(II)-insulin and Mg(II)-insulin, the formation equilibrium constants (Kf1) are in order of Ca(II)-insulin>Mg(II)-insulin>Na(I)-insulin>K(I)-insulin in water. The results showed that Na(I) and K(I) ions are involved in the formation of the non-covalent complexes with insulin molecule, since high extracellular and intracellular concentrations of them in the body.