Kyoung-Soon Jang

Multi-spectrometric analyses of lipoteichoic acids isolated from Lactobacillus plantarum

Lipoteichoic acid is a major cell wall virulence factor of gram-positive bacteria. LTAs from various bacteria have differential immunostimulatory potentials due to heterogeneity in their structures. Although recent studies have demonstrated that LTA isolated from Lactobacillus plantarum (pLTA) has anti-inflammatory properties and is less inflammatory than LTAs from pathogenic bacteria, little is known about the structure of pLTA. In this study, high-field NMR spectra of the pLTA were compared with those of LTA from pathogenic bacterium, Staphylococcus aureus (aLTA). The 2D NMR results demonstrated that pLTA possesses α-linked hexose sugar substituents on the poly-glycerophosphate backbone instead of N-acetylglucosamine substituents, and unsaturated fatty acids in its glycolipids. The sugar substituents were revealed as an approximately 29:1 molar ratio of the glucose to galactose by HPAEC-PAD analysis. MALDI-TOF/TOF MS analyses identified the presence of unsaturated fatty acids in the glycolipid moieties of pLTA. In addition, the glycolipid structure was found to be composed of trihexosyl-diacyl- and/or trihexosyl-triacyl-glycerol ceramide units by means of unique fragment ions of the glycolipids. These results enabled us to elucidate the pLTA structure, which is distinctively different from canonical LTA structure, and suggest that the unique immunological property of pLTA might be caused by the pLTA structure.

Rapid and high-throughput analysis of N-glycans from ovarian cancer serum using a 96-well plate platform

We present a rapid and high-throughput human serum N-glycan preparation technology using 96-well plate-based procedures. The released N-glycans from polyvinylidene fluoride (PVDF) membrane filter plate are subsequently loaded to porous graphitic carbon (PGC) containing a 96-well plate to remove salts and other contaminants without sacrificing accuracy or reproducibility. This robust glycan preparation technology is applied to ovarian cancer diagnosis using 5 microl of patient serum.

Mass spectrometric quantification of neutral and sialylated N-glycans from a recombinant therapeutic glycoprotein produced in the two Chinese hamster ovary cell lines.

Quality control and assurance of glycan profiles of a recombinant glycoprotein from lot to lot is a critical issue in the pharmaceutical industry. To develop an easy and simple quantitative and qualitative glycan profile method based on matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), the modification with Girards reagent T (GT) was exploited. Because GT-derivatized quantification of oligosaccharides using MALDI-TOF MS is possible only with neutral glycans, sialylated glycans are not subjected to quantitative analysis with MALDI-TOF MS. To solve this problem, mild methyl esterification and subsequent GT derivatization were employed, enabling us to perform rapid qualitative and quantitative analysis of sialylated and neutral N-linked oligosaccharides using MALDI-TOF MS. This modified method was used in the comparative quantification of N-glycans from the recombinant therapeutic glycoprotein expressed in two different Chinese hamster ovary (CHO) cell lines. The percentages of sialylated N-glycans to total were 22.5 and 5.2% in CHO-I and CHO-II cells, respectively, resulting in a significant difference in the biological activity of the recombinant glycoprotein.

Calcium Hydroxide Inactivates Lipoteichoic Acid from Enterococcus faecalis through Deacylation of the Lipid Moiety

INTRODUCTION Lipoteichoic acid (LTA) is a major virulence factor of Enterococcus faecalis that is closely associated with refractory apical periodontitis. Recently, we have shown that calcium hydroxide, a commonly used intracanal medicament, abrogated the ability of LTA to stimulate the production of tumor necrosis factor α in a murine macrophage line, RAW 264.7. Because calcium hydroxide could potentially modify the glycolipid moiety of LTA, we examined if calcium hydroxide inactivates LTA through deacylation of the LTA. METHODS LTA was prepared from E. faecalis by organic solvent extraction followed by chromatography with the hydrophobic-interaction column and the ion-exchange column. RAW 264.7 cells were stimulated with intact LTA or calcium hydroxide-treated LTA for 24 hours, and the productions of nitric oxide (NO) and chemokines interferon-gamma-induced protein (IP-10) and macrophage inflammatory protein-1α (MIP-1α) were determined. The glycolipid structure of LTA was analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry and thin layer chromatography (TLC). RESULTS The production of NO, IP-10, and MIP-1α was augmented in LTA-stimulated cells, whereas no such effect was observed upon stimulation with calcium hydroxide-pretreated LTA. Mass spectrometry showed that intact glycolipids of LTA yielded distinct mass peaks at 930 to 1,070 mass over charge (m/z) units, corresponding to dihexosyl-diacylglycerol consisting of two acyl chains with chain lengths of C(16) to C(22) and with one or two unsaturated double bonds. However, those peaks were not observed in the mass spectra of the calcium hydroxide-treated LTA. Furthermore, free fatty acids released from the calcium hydroxide-treated LTA were detected using TLC. CONCLUSION We suggest that calcium hydroxide attenuates the inflammatory activity of E. faecalis LTA through deacylation of the LTA.

Identification of staphylococcal lipoteichoic acid-binding proteins in human serum by high-resolution LTQ-Orbitrap mass spectrometry.

Lipoteichoic acid (LTA), a major virulence factor of Gram-positive bacteria, is associated with bacterial adherence to host cells, biofilm formation, and inflammation. LTA-binding proteins (LTA-BPs) play an important role in the host immune response by initially recognizing and responding to LTA during infections. In this study, we screened for LTA-BPs in human serum using LTA-immobilized beads and high-throughput mass spectrometry. Highly pure and structurally intact LTA was prepared from Staphylococcus aureus and immobilized onto N-hydroxysuccinimide-activated Sepharose(®) 4 Fast Flow beads. The immobilization process does not seem to affect the biological activity of LTA since LTA-immobilized beads could stimulate macrophages and activate Toll-like receptor 2. Then, the LTA-immobilized beads were incubated with the human serum to capture LTA-BPs and their molecular identities were determined using high-resolution LTQ-Orbitrap hybrid Fourier transform mass spectrometry. LTA-BPs captured at high frequencies were neutrophil-activating peptide 2, prohibitin-2, alpha-1-anti-trypsin, histidine-rich glycoprotein, apolipoproteins, complements, and coagulation factor, most of which are known to be related with the host immune responses against infections. As high-throughput, efficient, accurate and sensitive, this screening method could be widely applicable to the identification of novel binding proteins to microbial virulence factors with glycolipid structures.

Low mass cutoff evasion with qz value optimization in ion trap

An ion trap is a powerful analyzer because of its high resolution, high sensitivity, and multistage mass analysis (MS(n)) capabilities. Multiple fragmentation analysis provides useful information regarding peptide sequence and biomolecular structure; however, this approach is limited by an inherent low mass cutoff (LMCO) derived from collision-induced dissociation (CID). To avoid the LMCO for application of an ion trap to iTRAQ, we optimized the q(z) value, which is a parameter that is proportional to the applied fundamental AC radio frequency voltage of a tandem mass spectrometry (MS/MS) event. Considering that many ion trap MS analyses employ CID as the MS/MS method, this method can be a practical one without any instrumental changes.

A MALDI-MS-based quantitative analytical method for endogenous estrone in human breast cancer cells

The level of endogenous estrone, one of the three major naturally occurring estrogens, has a significant correlation with the incidence of post-menopausal breast cancer. However, it is challenging to quantitatively monitor it owing to its low abundance. Here, we develop a robust and highly sensitive mass-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based quantitative platform to identify the absolute quantities of endogenous estrones in a variety of clinical specimens. The one-step modification of endogenous estrone provided good linearity (R2 > 0.99) and significantly increased the sensitivity of the platform (limit of quantitation: 11 fmol). In addition, we could identify the absolute amount of endogenous estrones in cells of the breast cancer cell line MCF-7 (34 fmol/106 cells) by using a deuterated estrone as an internal standard. Finally, by applying the MALDI-MS-based quantitative method to endogenous estrones, we successfully monitored changes in the metabolic expression level of estrones (17.7 fmol/106 letrozole-treated cells) in MCF-7 cells resulting from treatment with an aromatase inhibitor. Taken together, these results suggest that this MALDI-MS-based quantitative approach may be a general method for the targeted metabolomics of ketone-containing metabolites, which can reflect clinical conditions and pathogenic mechanisms.

Rapid functional identification of putative genes based on the combined in vitro protein synthesis with mass spectrometry : A tool for functional genomics

For the rapid identification of functional activity of unknown genes from a sequence database, a new method based on in vitro protein synthesis combined with mass spectrometry was developed. To discriminate their subtle enzymatic activity, in vitro synthesized and one-step purified lipolytic enzymes, such as lip A and lip B from Bacillus subtilis and an unknown protein ybfF from Escherichia coli, were reacted with a mixture of triglycerides with different carbon chain lengths. Using direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of reaction product, all three enzymes were revealed to have strong esterase activity rather than true lipase activity, which has no reactivity on long-chain fatty acids such as triolein. These results were also confirmed by classical color assay using p-nitrophenyl butyrate (pNPB) and p-nitrophenyl palmitate (pNPP) as representative lipolytic substrates.

The sweets standing at the borderline between allo‐ and xenotransplantation

Animal cells are densely covered with glycoconjugates, such as N‐glycan, O‐glycan, and glycosphingolipids, which are important for various biological and immunological events at the cell surface and in the extracellular matrix. Endothelial α‐Gal carbohydrate epitopes (Galα3Gal‐R) expressed on porcine tissue or cell surfaces are such glycoconjugates and directly mediate hyperacute immunological rejection in pig‐to‐human xenotransplantation. Although researchers have been able to develop α1,3‐galactosyltransferase (GalT) gene knockout (KO) pigs, there remain unclarified non‐Gal antigens that prevent xenotransplantation. Based on our expertise in the structural analysis of xenoantigenic carbohydrates, we describe the immunologically significant non‐human carbohydrate antigens, including α‐Gal antigens, analyzed as part of efforts to assess the antigens responsible for hyperacute immunological rejection in pig‐to‐human xenotransplantation. The importance of studying human, pig, and GalT‐KO pig glycoprofiles, and of developing adequate pig‐to‐human glycan databases, is also discussed.

The Xeno-glycomics database (XDB): a relational database of qualitative and quantitative pig glycome repertoire

SUMMARY In recent years, the improvement of mass spectrometry-based glycomics techniques (i.e. highly sensitive, quantitative and high-throughput analytical tools) has enabled us to obtain a large dataset of glycans. Here we present a database named Xeno-glycomics database (XDB) that contains cell- or tissue-specific pig glycomes analyzed with mass spectrometry-based techniques, including a comprehensive pig glycan information on chemical structures, mass values, types and relative quantities. It was designed as a user-friendly web-based interface that allows users to query the database according to pig tissue/cell types or glycan masses. This database will contribute in providing qualitative and quantitative information on glycomes characterized from various pig cells/organs in xenotransplantation and might eventually provide new targets in the α1,3-galactosyltransferase gene-knock out pigs era. AVAILABILITY The database can be accessed on the web at http://bioinformatics.snu.ac.kr/xdb.