Cheol-Hwan Hwang

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.

Quantitative targeted metabolomics for 15d-deoxy-Δ12, 14-PGJ2 (15d-PGJ2) by MALDI-MS

Prostaglandins (PGs) are lipid mediators that may play important roles in cancer, immunomodulation, and neurodegeneration. So, the quantitative analysis of PGs will therefore be important in order to understand the natural history of a range of diseases and may be used as a tool in the development of new biotherapeutics. However, such an analysis is problematic because of the small quantities of PGs present in the body. Here, we developed a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS)-based analytical method for rapid and easy quantification of the ketone-containing PGs (15d-PGJ2etc.) as a targeted metabolomics platform. The chemical derivatization with Girard’s reagent P (GP) provided a good linearity (R2 > 0.99) between peak area and quantity of 15d-PGJ2 and highly improved sensitivity (limit of quantitation, LOQ: 1.25 pmol on a spot in MALDI plate). Finally, we utilized this method to directly characterize the interaction between peroxisome proliferatoractivated receptor gamma-ligand binding domain (PPARγ-LBD) and 15d-PGJ2. The 15d-PGJ2 was enriched by PPARγ-LBD and also the binding level of the ligand was dropped considerably by the treatment of PPARγ agonist such as rosiglitazone (about 0.61-fold reduction). Taken together, our MALDI-MS-based targeted metabolomics method for ketone-containing PGs may be applicable to elucidate the protein-metabolite interactions and to identify natural ligands for drug candidates.

Quantitative characterization of intact sialylated O-glycans with MALDI-MS for protein biotherapeutics

For validating O-glycosylation of protein biotherapeutics, we presented a quantitative O-glycomics method which is based on the neutralization of sialic acids, the specific release of O-glycans, and the introduction of permanent positive charge followed by quantitative MALDI-MS analysis. This method shows excellent technical reproducibility, linearity and sensitivity. In addition, it enables the quantification of intact O-glycans with minimal degradation or loss of sialic acids on these glycans compared to a conventional HPLC-based method. We then applied this method to quantitatively characterize O-glycans present on Etanercept. The analysis showed the relative abundances of mono- and di-sialylated core 1 O-glycans - were 79.3±0.8% and 17.3±1.4%, respectively. This glycomics technology could allow for the reliable quantitative analysis of intact O-glycans from glycoproteins and may contribute to validation of O-glycosylation protein biotherapeutics in the pharmaceutical industry.

Chemical Structure of the Lipid A component of Pseudomonas sp. strain PAMC 28618 from Thawing Permafrost in Relation to Pathogenicity

Climate change causes permafrost thawing, and we are confronted with the unpredictable risk of newly discovered permafrost microbes that have disease-causing capabilities. Here, we first characterized the detailed chemical structure of the lipid A moiety from a Pseudomonas species that was isolated from thawing arctic permafrost using MALDI-based mass spectrometric approaches (i.e., MALDI-TOF MS and MALDI-QIT-TOF MSn). The MALDI multi-stage mass spectrometry (MS) analysis of lipid A extracted from the Pseudomonas sp. strain PAMC 28618 demonstrated that the hexaacyl lipid A ([M−H]− at m/z 1616.5) contains a glucosamine (GlcN) disaccharide backbone, two phosphates, four main acyl chains and two branched acyl chains. Moreover, the lipid A molecule–based structural activity relationship with other terrestrial Gram-negative bacteria indicated that strain PAMC 28618 has an identical lipid A structure with the mesophilic Pseudomonas cichorii which can cause rot disease in endive (Cichorium endivia) and that their bacterial toxicities were equivalent. Therefore, the overall lipid A validation process provides a general strategy for characterizing bacteria that have been isolated from arctic permafrost and analyzing their respective pathogenicities.

Determination of Lipid A Profile of Gram-Negative Bacteria from Arctic Soils Using Mass Spectrometric Approaches

For decades, the microorganisms in arctic soils have been newly discovered according to the climate change and global warming. In this study, the chemical structure of a lipid A molecule from Pseudomonas sp. strain PAMC 28615 which was newly discovered from arctic soils was characterized by mass spectrometric approaches such as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and MALDI multi-stage tandem mass spectrometry (MS). First, lipopolysaccharide (LPS) from Pseudomonas sp. strain PAMC 28615 was extracted and subsequently hydrolyzed to obtain the lipid A. The parent ion peak at m/z 1632 was determined by MALDI-TOF MS, which also can validate our lipid A purification method. For detailed structural determination, we performed the multiple-stage tandem mass analysis (MS 4 ) of the parent ion, and subsequently the abundant fragment ions in each MS stage are tested. The fragment ions in each MS stage were produced from the loss of phosphate groups and fatty acyl groups, which could be used to confirm the composition or the position of the lipid A components. Consequently, the mass spectrometry-based lipid A profiling method could provide the detail chemical structure of lipid A from the Pseudomonas sp. strain PAMC 28615 as an arctic bacterium from the frozen arctic soil.

A solid-phase screening method for identification of glycan-binding cells

Glycan immobilization and epitope recognition on a solid support is useful for functional glycoproteomics and analysis of protein-protein interactions. However, the lack of high-throughput experimental methods for the examination of cell-glycan interactions means that identifying glycan-binding cells remains a challenge. Herein, we describe a tool which enables the screening of glycanbinding cells that specifically recognize the glycan epitopes released from the membrane glycoproteins of cells and tissues. Biotinylated pig kidney N-glycans were immobilized on streptavidin-conjugated solid supports and then incubated with human immune cell lines. U937 cells discriminated between the α-galactose (α-Gal) and non-Gal pig N-glycans on the solid support. This screening strategy could aid exploration of cell-protein or cell-cell interactions.