Publications by authors named "Jeong Hee Moon"

62 Publications

Comparative study on formation of protein coronas under three different serum origins.

Biointerphases 2020 11 13;15(6):061002. Epub 2020 Nov 13.

Bioimaging Team, Safety Measurement Institute, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, South Korea.

Nanomaterials form a complex called "protein corona" by contacting with protein-containing biological fluids such as serum when they are exposed to physiological environments. The characteristics of these proteins, which are one of the substantial factors in cellular response, are affected by the interactions between the nanomaterials and the biological systems. Many studies have investigated the biological behaviors of nanomaterials by conducting experiments in vitro and in vivo; however, the origin of the biological materials used is rather inconsistent. This is due to the fact that the composition of the protein coronas may differ depending on the animal origin, not on the composition or size of the nanoparticles. The resulting differences in the composition of the protein coronas can lead to different conclusions. To identify the differences in protein corona formation among sera of different species, we investigated protein coronas of gold and silica nanoparticles in serum obtained from various species. Using comparative proteomic analysis, common proteins adsorbed onto each nanoparticle among the three different sera were identified as highly abundant proteins in the serum. These findings indicate that protein corona formation is dependent on the serum population rather than the size or type of the nanoparticles. Additionally, in the physiological classification of protein coronas, human serum (HS) was found to be rich in apolipoproteins. In conclusion, our data indicate that HS components are different from those of bovine or mouse, indicating that the serum species origin should be carefully considered when selecting a biological fluid.
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http://dx.doi.org/10.1116/6.0000396DOI Listing
November 2020

Numerical evaluation of polyethylene glycol ligand conjugation to gold nanoparticle surface using ToF-SIMS and statistical analysis.

Biointerphases 2020 05 27;15(3):031008. Epub 2020 May 27.

Center for Nano-Bio Measurement, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, South Korea.

Nanoparticles (NPs) are substances between 1 and 100 nm in size. They have been the subject of numerous studies because of their potential applications in a wide range of fields such as cosmetics, electronics, medicine, and food. For biological applications of nanoparticles, they are usually coated with a substance capable of preventing agglomeration of the nanoparticles and nonspecific binding and exhibiting water-solubility characteristics with specific immobilized (bio)molecules. In order to evaluate the chemical properties of the surface-modified nanoparticles for bioapplications, including drug delivery, a simple and reliable method for the analysis of the presence of the surface chemicals and the ligand states of the nanoparticles is necessary. In this study, the authors numerically evaluated the extent of polyethylene glycol (PEG) ligand conjugation on AuNPs by concurrently adopting a microliquid inkjet printing system for sampling of the PEGylated AuNPs solution and ToF-SIMS imaging together with statistical analysis. The statistical correlation values calculated from the signals of PEG and Au measured by ToF-SIMS imaging on the sample spots made by a microliquid inkjet printing system showed better reproducibility and improved correlation values compared to the pipet spotting. Their improved method will be useful to evaluate ligand-conjugated nanoparticles for quality control of each conjugation process.
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http://dx.doi.org/10.1116/6.0000106DOI Listing
May 2020

Ar-gas cluster ion beam in ToF-SIMS for peptide and protein analysis.

Biointerphases 2020 04 14;15(2):021011. Epub 2020 Apr 14.

Center for Nano-Bio Measurement, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, South Korea.

Since Ar-gas cluster ion beams (Ar-GCIBs) have been introduced into time-of-flight secondary ion mass spectrometry (ToF-SIMS), there have been various attempts to analyze organic materials and biomolecules that require low-damage analysis and high sensitivity, because Ar-GCIBs allow soft ionization of large molecules such as peptides and proteins due to the low energy per atom. Here, the authors adopted the Ar-GCIB as a primary beam to detect proteins including human insulin, ubiquitin, and cytochrome C (molecular weights are 5808, 8564, and 12 327 Da, respectively). They have confirmed that the detection of the intact proteins was possible when the Ar-GCIB was used as a primary ion beam. In addition, they successfully identified each protein by analyzing the trypsin-digested peptides in myoglobin, cytochrome C, and bovine serum albumin. They also attempted on-surface enzymatic digestion to identify proteins on the surface of the Si wafer and obtained results identical to those of in-solution digestion. It is expected that the authors' on-surface digestion method can enable the application of ToF-SIMS for the analysis of proteins present in biological tissues.
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http://dx.doi.org/10.1116/6.0000105DOI Listing
April 2020

Quantitative proteomic analyses reveal that GPX4 downregulation during myocardial infarction contributes to ferroptosis in cardiomyocytes.

Cell Death Dis 2019 11 4;10(11):835. Epub 2019 Nov 4.

Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Korea.

Ischaemic heart disease (IHD) is the leading cause of death worldwide. Although myocardial cell death plays a significant role in myocardial infarction (MI), its underlying mechanism remains to be elucidated. To understand the progression of MI and identify potential therapeutic targets, we performed tandem mass tag (TMT)-based quantitative proteomic analysis using an MI mouse model. Gene ontology (GO) analysis and gene set enrichment analysis (GSEA) revealed that the glutathione metabolic pathway and reactive oxygen species (ROS) pathway were significantly downregulated during MI. In particular, glutathione peroxidase 4 (GPX4), which protects cells from ferroptosis (an iron-dependent programme of regulated necrosis), was downregulated in the early and middle stages of MI. RNA-seq and qRT-PCR analyses suggested that GPX4 downregulation occurred at the transcriptional level. Depletion or inhibition of GPX4 using specific siRNA or the chemical inhibitor RSL3, respectively, resulted in the accumulation of lipid peroxide, leading to cell death by ferroptosis in H9c2 cardiomyoblasts. Although neonatal rat ventricular myocytes (NRVMs) were less sensitive to GPX4 inhibition than H9c2 cells, NRVMs rapidly underwent ferroptosis in response to GPX4 inhibition under cysteine deprivation. Our study suggests that downregulation of GPX4 during MI contributes to ferroptotic cell death in cardiomyocytes upon metabolic stress such as cysteine deprivation.
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http://dx.doi.org/10.1038/s41419-019-2061-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828761PMC
November 2019

Covalent binding of uracil DNA glycosylase UdgX to abasic DNA upon uracil excision.

Nat Chem Biol 2019 06 17;15(6):607-614. Epub 2019 May 17.

Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.

Uracil DNA glycosylases (UDGs) are important DNA repair enzymes that excise uracil from DNA, yielding an abasic site. Recently, UdgX, an unconventional UDG with extremely tight binding to DNA containing uracil, was discovered. The structure of UdgX from Mycobacterium smegmatis in complex with DNA shows an overall similarity to that of family 4 UDGs except for a protruding loop at the entrance of the uracil-binding pocket. Surprisingly, H109 in the loop was found to make a covalent bond to the abasic site to form a stable intermediate, while the excised uracil remained in the pocket of the active site. H109 functions as a nucleophile to attack the oxocarbenium ion, substituting for the catalytic water molecule found in other UDGs. To our knowledge, this change from a catalytic water attack to a direct nucleophilic attack by the histidine residue is unprecedented. UdgX utilizes a unique mechanism of protecting cytotoxic abasic sites from exposure to the cellular environment.
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http://dx.doi.org/10.1038/s41589-019-0289-3DOI Listing
June 2019

Molecular-Level Understanding of Continuous Growth from Iron-Oxo Clusters to Iron Oxide Nanoparticles.

J Am Chem Soc 2019 05 18;141(17):7037-7045. Epub 2019 Apr 18.

Center for Nanoparticle Research , Institute for Basic Science (IBS) , Seoul 08826 , Republic of Korea.

The formation of inorganic nanoparticles has been understood based on the classical crystallization theory described by a burst of nucleation, where surface energy is known to play a critical role, and a diffusion-controlled growth process. However, this nucleation and growth model may not be universally applicable to the entire nanoparticle systems because different precursors and surface ligands are used during their synthesis. Their intrinsic chemical reactivity can lead to a formation pathway that deviates from a classical nucleation and growth model. The formation of metal oxide nanoparticles is one such case because of several distinct chemical aspects during their synthesis. Typical carboxylate surface ligands, which are often employed in the synthesis of oxide nanoparticles, tend to continuously remain on the surface of the nanoparticles throughout the growth process. They can also act as an oxygen source during the growth of metal oxide nanoparticles. Carboxylates are prone to chemical reactions with different chemical species in the synthesis such as alcohol or amine. Such reactions can frequently leave reactive hydroxyl groups on the surface. Herein, we track the entire growth process of iron oxide nanoparticles synthesized from conventional iron precursors, iron-oleate complexes, with strongly chelating carboxylate moieties. Mass spectrometry studies reveal that the iron-oleate precursor is a cluster comprising a tri-iron-oxo core and carboxylate ligands rather than a mononuclear complex. A combinatorial analysis shows that the entire growth, regulated by organic reactions of chelating ligands, is continuous without a discrete nucleation step.
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http://dx.doi.org/10.1021/jacs.9b01670DOI Listing
May 2019

Benproperine, an ARPC2 inhibitor, suppresses cancer cell migration and tumor metastasis.

Biochem Pharmacol 2019 05 30;163:46-59. Epub 2019 Jan 30.

Laboratory of Chemical Biology and Genomics, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahakro, Daejeon 34141, Republic of Korea; Korea University of Science and Technology in Korea, Daejeon, Republic of Korea. Electronic address:

Metastasis is the leading cause of cancer mortality and cancer cell migration is an essential stage of metastasis. We identified benproperine (Benp, a clinically used antitussive drug) as an inhibitor of cancer cell migration and an anti-metastatic agent. Benp selectively inhibited cancer cell migration and invasion, which also suppressed metastasis of cancer cells in animal models. Actin-related protein 2/3 complex subunit 2 (ARPC2) was identified as a molecular target of Benp by affinity column chromatography with Benp-tagged Sepharose beads. Benp bound directly to ARPC2 in cells, which was validated by pull-down assay using Benp-biotin and label-free biochemical methods such as the drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA). Benp inhibited Arp2/3 function, showing disruption of lamellipodial structure and inhibition of actin polymerization. Unlike Arp2/3 inhibitors, Benp selectively inhibited the migration of cancer cells but not normal cells. ARPC2-knockdown cancer cells showed defective cell migration and suppressed metastasis in an animal model. Therefore, ARPC2 is a potential target for anti-metastatic therapy, and Benp has the clinical potential to block metastasis. Furthermore, Benp is a useful agent for studying the functions of the Arp2/3 complex in cancer cell migration and metastasis.
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http://dx.doi.org/10.1016/j.bcp.2019.01.017DOI Listing
May 2019

2'-hydroxycinnamaldehyde inhibits cancer cell proliferation and tumor growth by targeting the pyruvate kinase M2.

Cancer Lett 2018 10 18;434:42-55. Epub 2018 Jul 18.

Laboratory of Chemical Biology and Genomics, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahakro, Daejeon, 34141, Republic of Korea; Korea University of Science and Technology in Korea, Daejeon, 34113, Republic of Korea. Electronic address:

It is reported that 2'-hydroxycinnamaldehyde (HCA), isolated from cinnamon, has anti-tumor effects through the modulation of multi-target molecules. In this study, we identified pyruvate kinase M2 (PKM2) as a direct target of HCA by use of biochemical methods including affinity chromatography, drug affinity responsive target stability, and cellular thermal shift assay. PKM2 is up-regulated in multiple cancer types and is considered as a potential target for cancer therapy. HCA binds directly to PKM2 and selectively decreases the phosphorylation of PKM2 at Tyr105, indicating a potential anti-proliferative effect on prostate cancer cells. As a PKM2 activator, HCA increases pyruvate kinase activity by promoting the tetrameric state of PKM2. However, HCA suppresses protein kinase activity of PKM2 by decreasing the phosphorylation at Tyr105. Moreover, this leads to a decrease of PKM2-mediated STAT3 phosphorylation at Tyr705 and a down-regulation of target genes, including MEK5 and cyclin D1. Furthermore, HCA suppresses tumor growth and the release of tumor extracellular vesicles in vivo by inhibiting the phosphorylation of PKM2. Collectively, our results suggest that HCA may be a potential anticancer agent targeting PKM2 in cancer progression.
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http://dx.doi.org/10.1016/j.canlet.2018.07.015DOI Listing
October 2018

Gain Switching for a Detection System to Accommodate a Newly Developed MALDI-Based Quantification Method.

J Am Soc Mass Spectrom 2017 09 10;28(9):1987-1990. Epub 2017 Jul 10.

Disease Target Structure Research Center, KRIBB, Daejeon, 305-806, Korea.

In matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), matrix-derived ions are routinely deflected away to avoid problems with ion detection. This, however, limits the use of a quantification method that utilizes the analyte-to-matrix ion abundance ratio. In this work, we will show that it is possible to measure this ratio by a minor instrumental modification of a simple form of MALDI-TOF. This involves detector gain switching. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-017-1711-2DOI Listing
September 2017

Structural and mechanistic insights into the inhibition of class C β-lactamases through the adenylylation of the nucleophilic serine.

J Antimicrob Chemother 2017 03;72(3):735-743

Department of Chemistry and Nano Science, Ewha Womans University, Seoul, 03760, Republic of Korea.

Objectives: : Investigation into the adenylylation of the nucleophilic serine in AmpC BER and CMY-10 extended-spectrum class C β-lactamases.

Methods: : The formation and the stability of the adenylate adduct were examined by X-ray crystallography and MS. Inhibition assays for kinetic parameters were performed by monitoring the hydrolytic activity of AmpC BER and CMY-10 using nitrocefin as a reporter substrate. The effect of adenosine 5'-(P-acetyl)monophosphate (acAMP) on the MIC of ceftazidime was tested with four Gram-negative clinical isolates.

Results: : The crystal structures and MS analyses confirmed the acAMP-mediated adenylylation of the nucleophilic serine in AmpC BER and CMY-10. acAMP inhibited AmpC BER and CMY-10 through the adenylylation of the nucleophilic serine, which could be modelled as a two-step mechanism. The initial non-covalent binding of acAMP to the active site is followed by the covalent attachment of its AMP moiety to the nucleophilic serine. The inhibition efficiencies ( k inact / K I ) of acAMP against AmpC BER and CMY-10 were determined to be 320 and 140 M -1  s -1 , respectively. The combination of ceftazidime and acAMP reduced the MIC of ceftazidime against the tested bacteria.

Conclusions: : Our structural and kinetic studies revealed the detailed mechanism of adenylylation of the nucleophilic serine and may serve as a starting point for the design of novel class C β-lactamase inhibitors on the basis of the nucleotide scaffold.
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http://dx.doi.org/10.1093/jac/dkw491DOI Listing
March 2017

Quantification of Carbohydrates and Related Materials Using Sodium Ion Adducts Produced by Matrix-Assisted Laser Desorption Ionization.

J Am Soc Mass Spectrom 2016 11 19;27(11):1887-1890. Epub 2016 Sep 19.

Seoul National University Research Institute for Basic Sciences, Seoul, 151-747, Korea.

The utility of sodium ion adducts produced by matrix-assisted laser desorption ionization for the quantification of analytes with multiple oxygen atoms was evaluated. Uses of homogeneous solid samples and temperature control allowed the acquisition of reproducible spectra. The method resulted in a direct proportionality between the ion abundance ratio I([A + Na])/I([M + Na]) and the analyte concentration, which could be used as a calibration curve. This was demonstrated for carbohydrates, glycans, and polyether diols with dynamic range exceeding three orders of magnitude. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-016-1495-9DOI Listing
November 2016

Quantitative transfer of polar analytes on a solid surface to a liquid matrix in MALDI profiling.

J Mass Spectrom 2016 Dec;51(12):1152-1156

Seoul National University Research Institute for Basic Sciences, Seoul, 151-747, Korea.

In profiling of a specimen by matrix-assisted laser desorption ionization (MALDI) using a solid matrix, the solvent of the matrix solution extracts an analyte(s). A quantitative profiling cannot be achieved if the solvent evaporates before the complete extraction of the analyte. The extraction can become more quantitative when a liquid matrix dissolved in a solvent is used, which remains a liquid even after the evaporation of the solvent. To check this, radii of an analyte circle (r ), a matrix solution drop (r ) and a liquid matrix (r ) remaining after the solvent evaporation were controlled. Three types of samples were prepared, case A (r , r  < r ), case B (r  ≤ r  < r ) and case C (r  < r  < r ). In case A, the analyte amount in the matrix layer determined by MALDI was the same as the prepared amount inside the analyte circle. In case B, the analyte amount was the same as the amount inside the matrix circle. Only the analytes in contact with the liquid matrix layer, not more and not less, are transferred to the matrix layer. In case C, the analyte amount was greater than the amount inside the matrix circle, presumably because some of the analyte outside the matrix circle was dissolved by the solvent of the matrix solution. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/jms.3886DOI Listing
December 2016

Covalent and Oriented Surface Immobilization of Antibody Using Photoactivatable Antibody Fc-Binding Protein Expressed in Escherichia coli.

Anal Chem 2016 10 14;88(19):9503-9509. Epub 2016 Sep 14.

Hazards Monitoring Bionano Research Center, ‡Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, and §Department of Nanobiotechnology, University of Science and Technology , 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.

Fc-specific antibody binding proteins (FcBPs) with the minimal domain of protein G are widely used for immobilization of well-oriented antibodies onto solid surfaces, but the noncovalently bound antibodies to FcBPs are unstable in sera containing large amounts of antibodies. Here we report novel photoactivatable FcBPs with photomethionine (pMet) expressed in E. coli, which induce Fc-specific photo-cross-linking with antibodies upon UV irradiation. Unfortunately, pMet did not support protein expression in the native E. coli system, and therefore we also developed an engineered methionyl tRNA synthetase (MRS5m). Coexpression of MRS5m proteins successfully induced photoactivatable FcBP overexpression in methionine-auxotroph E. coli cells. The photoactivatable FcBPs could be easily immobilized on beads and slides via their N-terminal cysteine residues and 6xHis tag. The antibodies photo-cross-linked onto the photoactivatable FcBP-beads were resistant from serum-antibody mediated dissociation and efficiently captured antigens in human sera. Furthermore, photo-cross-linked antibody arrays prepared using this system allowed sensitive detection of antigens in human sera by sandwich immunoassay. The photoactivatable FcBPs will be widely applicable for well-oriented antibody immobilization on various surfaces of microfluidic chips, glass slides, and nanobeads, which are required for development of sensitive immunosensors.
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http://dx.doi.org/10.1021/acs.analchem.6b02071DOI Listing
October 2016

Improved mass resolution and mass accuracy in TOF-SIMS spectra and images using argon gas cluster ion beams.

Biointerphases 2016 Jun 9;11(2):02A321. Epub 2016 Jun 9.

Center for Nano-Bio Measurement, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, South Korea and Department of Nano Science, University of Science and Technology, Daejeon 34113, South Korea.

The popularity of argon gas cluster ion beams (Ar-GCIB) as primary ion beams in time-of-flight secondary ion mass spectrometry (TOF-SIMS) has increased because the molecular ions of large organic- and biomolecules can be detected with less damage to the sample surfaces. However, Ar-GCIB is limited by poor mass resolution as well as poor mass accuracy. The inferior quality of the mass resolution in a TOF-SIMS spectrum obtained by using Ar-GCIB compared to the one obtained by a bismuth liquid metal cluster ion beam and others makes it difficult to identify unknown peaks because of the mass interference from the neighboring peaks. However, in this study, the authors demonstrate improved mass resolution in TOF-SIMS using Ar-GCIB through the delayed extraction of secondary ions, a method typically used in TOF mass spectrometry to increase mass resolution. As for poor mass accuracy, although mass calibration using internal peaks with low mass such as hydrogen and carbon is a common approach in TOF-SIMS, it is unsuited to the present study because of the disappearance of the low-mass peaks in the delayed extraction mode. To resolve this issue, external mass calibration, another regularly used method in TOF-MS, was adapted to enhance mass accuracy in the spectrum and image generated by TOF-SIMS using Ar-GCIB in the delayed extraction mode. By producing spectra analyses of a peptide mixture and bovine serum albumin protein digested with trypsin, along with image analyses of rat brain samples, the authors demonstrate for the first time the enhancement of mass resolution and mass accuracy for the purpose of analyzing large biomolecules in TOF-SIMS using Ar-GCIB through the use of delayed extraction and external mass calibration.
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http://dx.doi.org/10.1116/1.4941447DOI Listing
June 2016

Discovery of a solvent effect preventing quantitative profiling by matrix-assisted laser desorption/ionization and its treatment.

Rapid Commun Mass Spectrom 2016 Feb;30(3):386-92

Seoul National University Research Institute for Basic Sciences, Seoul, 151-747, Korea.

Rationale: In analyte profiling by matrix-assisted laser desorption/ionization (MALDI), drawing a quantitative profile map is an outstanding problem. Recently, we developed a method to quantify an analyte by MALDI, which is needed to solve the problem. Another requirement for quantitative profiling is the quantitative sample-to-matrix analyte transfer, which is investigated in this work.

Methods: MALDI-time-of-flight (TOF) spectra were acquired for samples produced by two methods. In one, a sample solution containing a matrix and an analyte was loaded with a pipet and dried. In the other, a sample was prepared by a consecutive process, i.e., loading-drying of an analyte solution followed by that of a matrix solution. Two different micro-spotters were used in the second method. Various mixtures of organic solvents with water were used to prepare matrix solutions.

Results: The organic solvent, matrix, and analyte used in the study did not affect the analyte transfer efficiency, whereas it improved as the water content in the solvent increased. It also improved as the liquid droplet emitted by a micro-spotter got larger. Use of a more polar solvent or a larger droplet increases the contact time between a solution droplet and the sample surface, which seems to be responsible for the improvement in the transfer efficiency.

Conclusions: Sample-to-matrix analyte transfer occurred efficiently when polar solvents and/or large liquid droplets were used to produce solid samples for MALDI profiling with a micro-spotter. A long contact time between the sample surface and a matrix solution droplet is one of the requirements for quantitative profiling. Copyright © 2015 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/rcm.7452DOI Listing
February 2016

Acquisition of the depth profiles and reproducible mass spectra in matrix-assisted laser desorption/ionization of inhomogeneous samples.

Rapid Commun Mass Spectrom 2015 Apr;29(8):745-52

Department of Chemistry, Seoul National University, Seoul, 151-747, Korea.

Rationale: In our previous analysis of the matrix-assisted laser desorption/ionization (MALDI) spectra of peptides, we treated their depth profiles in solid samples as homogeneous. Here, we wanted to determine if the reproducible MALDI spectra and linear calibration curves reported previously would be obtained even when the depth profiles were inhomogeneous.

Methods: We derived a formula relating shot-number-dependent ion abundance data in temperature-controlled MALDI with the analyte depth profile in a solid sample. We prepared samples containing peptides, amino acids, and serotonin in α-cyano-4-hydroxycinnamic acid matrix by vacuum-drying and micro-spotting methods, recorded their MALDI spectra, and analyzed them with the aforementioned formula.

Results: For the samples prepared by vacuum-drying, the analyte depth profiles were inhomogeneous and maximized at the sample surface. Although the MALDI spectra changed as the shot continued, their sum over the entire set of spectra acquired from a spot was reproducible. Similarly, a high-quality calibration curve could be obtained with the spectral data summed over the entire set. Depth profiles were homogeneous for samples prepared by micro-spotting.

Conclusions: A method has been developed to obtain a reproducible MALDI spectrum and a linear calibration curve for an analyte with an inhomogeneous depth profile in a solid sample.
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http://dx.doi.org/10.1002/rcm.7157DOI Listing
April 2015

Investigations of Some Liquid Matrixes for Analyte Quantification by MALDI.

J Am Soc Mass Spectrom 2015 Oct 30;26(10):1657-64. Epub 2015 Jun 30.

Seoul National University Research Institute for Basic Sciences, Seoul, 151-747, Korea.

Sample inhomogeneity is one of the obstacles preventing the generation of reproducible mass spectra by MALDI and to their use for the purpose of analyte quantification. As a potential solution to this problem, we investigated MALDI with some liquid matrixes prepared by nonstoichiometric mixing of acids and bases. Out of 27 combinations of acids and bases, liquid matrixes could be produced from seven. When the overall spectral features were considered, two liquid matrixes using α-cyano-4-hydroxycinnamic acid as the acid and 3-aminoquinoline and N,N-diethylaniline as bases were the best choices. In our previous study of MALDI with solid matrixes, we found that three requirements had to be met for the generation of reproducible spectra and for analyte quantification: (1) controlling the temperature by fixing the total ion count, (2) plotting the analyte-to-matrix ion ratio versus the analyte concentration as the calibration curve, and (3) keeping the matrix suppression below a critical value. We found that the same requirements had to be met in MALDI with liquid matrixes as well. In particular, although the liquid matrixes tested here were homogeneous, they failed to display spot-to-spot spectral reproducibility unless the first requirement above was met. We also found that analyte-derived ions could not be produced efficiently by MALDI with the above liquid matrixes unless the analyte was sufficiently basic. In this sense, MALDI processes with solid and liquid matrixes should be regarded as complementary techniques rather than as competing ones.
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http://dx.doi.org/10.1007/s13361-015-1202-2DOI Listing
October 2015

Successful live birth after transfer of blastocyst and frozen blastocyst from rescue ICSI with application of polarized light microscopy for spindle examination on unfertilized eggs.

J Ovarian Res 2015 Apr 8;8:22. Epub 2015 Apr 8.

Department of Obstetrics and Gynaecology, MUHC Reproductive Center, McGill University Health Center (MUHC), McGill University, Montreal, QC, Canada.

This article aims to report successful live births after transfer of fresh blastocyst or vitrified/warmed blastocyst derived from intracytoplasmic sperm injection (ICSI) on day-1 of unfertilized mature eggs (so-called "rescue ICSI") with spindle examination using polarized light microscopy. Two couples who had rescue ICSI performed achieved a positive pregnancy result after the transfer of a fresh or vitrified blastocyst. The two pregnancies led to the live births of a healthy baby boy of 2.72 kg and baby girl of 3.4 kg, respectively.
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http://dx.doi.org/10.1186/s13048-015-0150-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4396738PMC
April 2015

Quick quantification of proteins by MALDI.

J Mass Spectrom 2015 Mar;50(3):596-602

Department of Chemistry, Seoul National University, Seoul, 151-747, Korea.

Previously, we reported that the matrix-assisted laser desorption ionization spectrum of a peptide became reproducible when an effective temperature was held constant. Using a calibration curve drawn by plotting the peptide-to-matrix ion abundance ratio versus the peptide concentration in a solid sample, a peptide could be quantified without the use of any internal standard. In this work, we quantified proteins by quantifying their tryptic peptides with the aforementioned method. We modified the digestion process; e.g. disulfide bonds were not cleaved, so that hardly any reagent other than trypsin remained after the digestion process. This allowed the preparation of a sample by the direct mixing of a digestion mixture with a matrix solution. We also observed that the efficiency of the matrix-to-peptide proton transfer, as measured by its reaction quotient, was similar for peptides with arginine at the C-terminus. With the reaction quotient averaged over many such peptides, we could rapidly quantify proteins. Most importantly, no peptide standard, not to mention its isotopically labeled analog, was needed in this method.
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http://dx.doi.org/10.1002/jms.3567DOI Listing
March 2015

A comparison of biochemical pregnancy rates between women who underwent IVF and fertile controls who conceived spontaneously†.

Hum Reprod 2015 Apr 11;30(4):783-8. Epub 2015 Feb 11.

Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, McGill University, 687 Pine Ave West, Montreal, Quebecc, Canada H3A 1A1

Study Question: Does IVF affect the biochemical pregnancy rate?

Summary Answer: The likelihood of an early pregnancy loss may be lower and is certainly not higher in IVF cycles when compared with published rates of biochemical pregnancy in fertile women ≤42 years old.

What Is Known Already: The use of gonadotrophins to stimulate multi-folliculogenesis alters endometrial expression of genes and proteins, compared with unstimulated cycles. Exogenous estrogen and progesterone taken for endometrial preparation in frozen embryo transfer cycles, also cause changes in endometrial gene and protein expression .These endometrial alterations may compromise the ability of embryos to develop once implanted, possibly increasing the biochemical pregnancy rate.

Study Design, Size, Duration: This is a retrospective study, involving 1636 fresh and 188 frozen, single embryo transfer (SET) IVF cycles performed between August 2008 and December 2012. The biochemical pregnancy rate of the 1824 combined IVF and frozen cycles were compared with fertile controls, derived from the three prospective studies in the medical literature that evaluate this rate.

Participants/materials, Setting, Methods: Subjects ≤42-years old, who underwent a SET, as part of a fresh or thawed IVF cycle were considered for inclusion. Each subject is represented only once. The biochemical pregnancy rates were compared with those of historical standard, fertile populations with spontaneous conceptions.

Main Results And The Role Of Chance: The pregnancy rates per transfer for fresh and frozen IVF cycles were similar at 39 and 40%, respectively. There was also no significant difference in the likelihood of pregnancy outcomes (clinical, biochemical and ectopic pregnancy) between fresh IVF and frozen cycles (85.4 versus 85.6%, 13.8 versus 14.8%, 0.5 versus 0%, P = 0.82). However, pregnancy rates decreased in older patients when compared with younger ones P < 0.0001. The biochemical pregnancy rate for fresh and frozen IVF cycles combined was 13.8% of all pregnancies. IVF and frozen cycles were combined as the IVF group treated with hormones for further comparison with the fertile control group. The biochemical pregnancy rate (14%) in the IVF group was lower than the rate based on the total fertile group (18%), P = 0.01 and differed significantly from the rate in two out of the three studies used to establish the normative rate. The age ranges of the IVF and fertile controls were 21-42 years. The mean age in the IVF population was 34.8 years, as compared with 29 years, 29, 4 years and 30.6 years (Zinaman) in the three published studies (mean: 29.4 years).

Limitations, Reasons For Caution: This is a retrospective study and it was impossible to recruit an in-house biochemical pregnancy control population.

Wider Implications Of The Findings: Lower early pregnancy wastage after IVF may be due to the opportunity to select the embryo for transfer. This finding should be confirmed in further studies but supports the idea that embryo selection is an important step.

Study Funding/competing Interests: None.
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http://dx.doi.org/10.1093/humrep/dev024DOI Listing
April 2015

Formation of gas-phase peptide ions and their dissociation in MALDI: insights from kinetic and ion yield studies.

Mass Spectrom Rev 2015 Mar-Apr;34(2):94-115. Epub 2014 May 26.

Medical Proteomics Research Center, KRIBB, Daejeon, 305-806, Korea.

Insights on mechanisms for the generation of gas-phase peptide ions and their dissociation in matrix-assisted laser desorption ionization (MALDI) gained from the kinetic and ion yield studies are presented. Even though the time-resolved photodissociation technique was initially used to determine the dissociation kinetics of peptide ions and their effective temperature, it was replaced by a simpler method utilizing dissociation yields from in-source decay (ISD) and post-source decay (PSD). The ion yields for a matrix and a peptide were measured by repeatedly irradiating a region on a sample and collecting ion signals until the sample in the region was completely depleted. Matrix- and peptide-derived gas-phase cations were found to be generated by pre-formed ion emission or by ion-pair emission followed by anion loss, but not by laser-induced ionization. The total number of ions, that is, matrix plus peptide, was found to be equal to the number of ions emitted from a pure matrix. A matrix plume was found to cool as it expanded, from around 800-1,000 K to 400-500 K. Dissociation of peptide ions along b/y channels was found to occur statistically, that is, following RRKM behavior. Small critical energy (E0  = 0.6-0.7 eV) and highly negative critical entropy (ΔS(‡)  = -30 to -25 eu) suggested that the transition structure was stabilized by multiple intramolecular interactions.
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http://dx.doi.org/10.1002/mas.21427DOI Listing
October 2015

Spectral reproducibility and quantification of peptides in MALDI of samples prepared by micro-spotting.

J Am Soc Mass Spectrom 2014 Aug 21;25(8):1502-5. Epub 2014 May 21.

Department of Chemistry, Seoul National University, Seoul, 151-747, Korea.

Previously, we reported that MALDI spectra of peptides became reproducible when temperature was kept constant. Linear calibration curves derived from such spectral data could be used for quantification. Homogeneity of samples was one of the requirements. Among the three popular matrices used in peptide MALDI [i.e., α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and sinapinic acid (SA)], homogeneous samples could be prepared by conventional means only for CHCA. In this work, we showed that sample preparation by micro-spotting improved the homogeneity for all three cases.
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http://dx.doi.org/10.1007/s13361-014-0919-7DOI Listing
August 2014

Dual track time-of-flight mass spectrometry for peptide quantification with matrix-assisted laser desorption/ionization.

Rapid Commun Mass Spectrom 2014 Apr;28(7):787-92

Department of Chemistry, Seoul National University, Seoul, 151-747, Korea.

Rationale: Previously, we reported a method (Anal. Chem. 2012, 84, 10332) for peptide quantification based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). In the method, the peptide-to-matrix ion abundance ratio was utilized. Implementation of the method with a commercial MALDI-TOF can be somewhat inconvenient because matrix-derived ions are routinely deflected away to avoid detector saturation. A solution for this inconvenience is required.

Methods: We installed a detector to acquire the TOF spectrum of the ions thrown away to avoid detector saturation. By sending the matrix- and peptide-derived ions along two different tracks and detecting them with different detectors, the inconvenience mentioned above could be avoided.

Results: Excellent linearity of the calibration curves obtained by the dual track TOF spectrometry is demonstrated. The method also allows for the acquisition of the tandem mass spectrum of a selected peptide, which can be useful for its identification.

Conclusions: We devised the dual track MALDI-TOF MS method to avoid detector saturation and demonstrated that the quantification and identification of peptides can be performed simultaneously.
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http://dx.doi.org/10.1002/rcm.6845DOI Listing
April 2014

Why do the abundances of ions generated by MALDI look thermally determined?

J Am Soc Mass Spectrom 2013 Nov 29;24(11):1807-15. Epub 2013 Aug 29.

Department of Chemistry, Seoul National University, Seoul, 151-747, Korea.

In a previous study (J. Mass Spectrom. 48, 299-305, 2013), we observed that the abundance of each ion in a matrix-assisted laser desorption ionization (MALDI) spectrum looked thermally determined. To find out the explanation for the phenomenon, we estimated the ionization efficiency and the reaction quotient (QA) for the autoprotolysis of matrix, M + M → [M + H](+) + [M - H](-), from the temperature-controlled laser desorption ionization spectra of α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). We also evaluated the equilibrium constants (KA) for the autoprotolysis at various temperatures by quantum chemical calculation. Primary ion formation via various thermal models followed by autoprotolysis-recombination was compatible with the observations. The upper limit of the effective temperature of the plume where autoprotolysis-recombination occurs was estimated by equating QA with the calculated equilibrium constant. Figure ᅟ
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http://dx.doi.org/10.1007/s13361-013-0717-7DOI Listing
November 2013

Matrix suppression as a guideline for reliable quantification of peptides by matrix-assisted laser desorption ionization.

Anal Chem 2013 Sep 29;85(18):8796-801. Epub 2013 Aug 29.

Department of Chemistry, Seoul National University , Seoul 151-742, Korea.

We propose to divide matrix suppression in matrix-assisted laser desorption ionization into two parts, normal and anomalous. In quantification of peptides, the normal effect can be accounted for by constructing the calibration curve in the form of peptide-to-matrix ion abundance ratio versus concentration. The anomalous effect forbids reliable quantification and is noticeable when matrix suppression is larger than 70%. With this 70% rule, matrix suppression becomes a guideline for reliable quantification, rather than a nuisance. A peptide in a complex mixture can be quantified even in the presence of large amounts of contaminants, as long as matrix suppression is below 70%. The theoretical basis for the quantification method using a peptide as an internal standard is presented together with its weaknesses. A systematic method to improve quantification of high concentration analytes has also been developed.
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http://dx.doi.org/10.1021/ac401967nDOI Listing
September 2013

Spindle examination in unfertilized eggs using the polarization microscope can assist rescue ICSI.

Reprod Biomed Online 2013 Mar 14;26(3):280-5. Epub 2012 Nov 14.

MUHC Reproductive Center, Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada.

Rescue ICSI can induce a high rate of 3 pronuclei (PN) formation from double insemination in eggs already fertilized by IVF but lacking signs of normal pronuclear formation. This study was performed to determine whether the number of 3PN embryos could be reduced by using the polarization microscope for rescue intracytoplasmic sperm injection (ICSI). As a study group, after conventional insemination, 81 unfertilized mature oocytes from 11 couples were checked for the number of spindles using the polarization microscope. One spindle (82.7%) or two spindles (17.3%) were observed in this group. Rescue ICSI was only performed on the unfertilized oocytes showing one spindle. In the control group, 87 mature oocytes which lacked visualization of any fertilization signs were selected for rescue ICSI and none of them underwent observation of the spindle. After rescue ICSI, the normal fertilization rate in the study group was significantly higher than in the control (68.7% versus 43.7%; P=0.0032). The rate of 3PN or 4PN embryos was significantly decreased in the study group with one spindle compared with the group without observation of the spindle (4.5% versus 26.4%; P=0.0004).
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http://dx.doi.org/10.1016/j.rbmo.2012.10.019DOI Listing
March 2013

Degree of ionization in MALDI of peptides: thermal explanation for the gas-phase ion formation.

J Am Soc Mass Spectrom 2012 Aug 1;23(8):1326-35. Epub 2012 Jun 1.

Department of Chemistry, Seoul National University, Seoul 151-742, Korea.

Degree of ionization (DI) in matrix-assisted laser desorption ionization (MALDI) was measured for five peptides using α-cyano-4-hydroxycinnanmic acid (CHCA) as the matrix. DIs were low 10(-4) for peptides and 10(-7) for CHCA. Total number of ions (i.e., peptide plus matrix) was the same regardless of peptides and their concentration, setting the number of gas-phase ions generated from a pure matrix as the upper limit to that of peptide ions. Positively charged cluster ions were too weak to support the ion formation via such ions. The total number of gas-phase ions generated by MALDI, and that from pure CHCA, was unaffected by the laser pulse energy, invalidating laser-induced ionization of matrix molecules as the mechanism for the primary ion formation. Instead, the excitation of matrix by laser is simply a way of supplying thermal energy to the sample. Accepting strong Coulomb attraction felt by cations in a solid sample, we propose three hypotheses for gas-phase peptide ion formation. In Hypothesis 1, they originate from the dielectrically screened peptide ions in the sample. In Hypothesis 2, the preformed peptide ions are released as part of neutral ion pairs, which generate gas-phase peptide ions via reaction with matrix-derived cations. In Hypothesis 3, neutral peptides released by ablation get protonated via reaction with matrix-derived cations.
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http://dx.doi.org/10.1007/s13361-012-0406-yDOI Listing
August 2012

Phosphoproteomic analysis of electroacupuncture analgesia in an inflammatory pain rat model.

Mol Med Rep 2012 Jul 18;6(1):157-62. Epub 2012 Apr 18.

Division of Meridian and Structural Medicine, School of Korean Medicine, Pusan National University, Yangsan 626-870, Republic of Korea.

The phosphorylation changes of nociceptive signaling proteins in the spinal cord dorsal horn (SCDH) are important in creating exaggerated pain following peripheral inflammation. Electroacupuncture (EA) has been widely used to relieve acute and chronic inflammatory pain in human and experimental pain models. In the present study, we performed a phosphoproteomic analysis to investigate whether EA alters protein phosphorylation in SCDH to attenuate pain development. Inflammatory hyperalgesia was induced by intraplantar injection of complete Freund's adjuvant (CFA) into the rat hind paw. EA treatment at ST36 and SP6 acupoints alleviated thermal hyperalgesia of the CFA-induced inflammatory pain model rats. The SCDH proteins from the control, inflammatory pain model and EA treatment rats were separated by 2-dimensional gel electrophoresis and the alterations in phosphoproteins were detected by Pro-Q Diamond staining. Eight proteins were differentially phosphorylated following EA treatment in the inflammatory pain model. Aldolase C, nascent polypeptide-associated complex α, stress-induced phosphoprotein 1 and heat shock protein 90 were identified as phosphoproteins whose expression was increased, whereas GDP dissociation inhibitor 1, thiamine triphosphatase, phosphoglycerate kinase 1 and 14-3-3 γ were phosphoproteins whose expression was decreased. This is the first phosphoproteomic screening study to elucidate the working mechanisms of EA analgesia. The results suggest that the regulation of cellular pathways in which the identified proteins are involved may be associated with an EA analgesic mechanism.
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http://dx.doi.org/10.3892/mmr.2012.879DOI Listing
July 2012

Ion yields for some salts in MALDI: mechanism for the gas-phase ion formation from preformed ions.

J Am Soc Mass Spectrom 2012 Jan 3;23(1):162-70. Epub 2011 Nov 3.

Medical Proteomics Research Center, KRIBB, Daejeon, 305-806, Korea.

Preformed ion emission is the main assumption in one of the prevailing theories for peptide and protein ion formation in matrix-assisted laser desorption ionization (MALDI). Since salts are in preformed ion forms in the matrix-analyte mixture, they are ideal systems to study the characteristics of preformed ion emission. In this work, a reliable method to measure the ion yield (IY) in MALDI was developed and used for a solid salt benzyltriphenylphosphonium chloride and two room-temperature ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate and trihexyltetradecylphosphonium bis(2,4,4-trimethylpentyl)phosphinate. IY for the matrix (α-cyano-4-hydroxycinnamic acid, CHCA) was also measured. Taking 1 pmol salts in 25 nmol CHCA as examples, IYs for three salts were similar, (4-8) × 10(-4), and those for CHCA were (0.8-1.2) × 10(-7). Even though IYs for the salts and CHCA remained virtually constant at low analyte concentration, they decreased as the salt concentrations increased. Two models, Model 1 and Model 2, were proposed to explain low IYs for the salts and the concentration dependences. Both models are based on the fact that the ion-pair formation equilibrium is highly shifted toward the neutral ion pair. In Model 1, the gas-phase analyte cations were proposed to originate from the same cations in the solid that were dielectrically screened from counter anions by matrix neutrals. In Model 2, preformed ions were assumed to be released from the solid sample in the form of neutral ion pairs and the anions in the ion pairs were assumed to be eliminated via reactions with matrix-derived cations.
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http://dx.doi.org/10.1007/s13361-011-0278-6DOI Listing
January 2012

Expansion cooling in the matrix plume is under-recognized in MALDI mass spectrometry.

J Am Soc Mass Spectrom 2011 Jun 9;22(6):1070-8. Epub 2011 Apr 9.

Department of Chemistry, Seoul National University, Seoul, Korea.

Time-of-flight (TOF) mass spectra for a peptide (Y(6)) were obtained by utilizing matrix-assisted infrared laser desorption ionization (IR-MALDI) with glycerol as the matrix and by ultraviolet MALDI with α-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), and 2,5-dihydroxybenzoic acid (DHB). Collisional activation during ion extraction and exothermicity in the gas-phase proton transfer were found to be unimportant as the driving forces for in-source (ISD) and post-source (PSD) decays, indicating that the thermal energy acquired during photo-ablation is responsible for their occurrence. The temperatures of [Y(6) + H](+) in the 'early' and 'late' matrix plumes were estimated by the kinetic analysis of the ISD and PSD yields, respectively. The order of the temperatures was glycerol < DHB ≈ SA < CHCA in the early plume and glycerol < DHB < SA < CHCA in the late plume. For each matrix, the temperature in the late plume was lower than in the early plume by 300-400 K, which was attributed to expansion cooling. The model (thermalization followed by expansion cooling) proposed to explain the occurrence of both rapid ISD and slow PSD is not only in sharp contrast with but also mutually exclusive with the prevailing explanation that the exothermicity in proton transfer and in-plume collisional activation are the driving forces for ion fragmentation in MALDI. The model also explains why MALDI is more successful for mass spectrometry of labile molecules than other desorption techniques that do not utilize a matrix. Factors affecting the plume temperature are also discussed.
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http://dx.doi.org/10.1007/s13361-011-0115-yDOI Listing
June 2011