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Se-Young Jang
Code / Date
YS-1 / March 30 (Fri) 08:00-08:06
Speaker
Se-Young Jang   CV
Affiliation
KIST
Title
A vinyl Sulfone-Based Fluorogenic Probe Capable of Selective Labeling of PHGDH in Live Mammalian Cells
Abstract

Quantitative chemical proteomics with activity-based probes is powerful tool to study various biological phenomena. However, because of complexity of biological systems, it is difficult to label specific proteins with small molecule in enough quantity for studying human diseases. Phosphoglycerate dehydrogenase (PHGDH) is a key enzyme in the biosynthesis of 3-phosphohydroxypyruvate which is first step in the serine biosynthesis pathway. Normaly cancer cells use the PHGDH and NAD to oxidise the 3-phosphoglycerate generated from glycolysis into the serine precursor 3-phosphohydroxypyruvate. Recent cancer research found melanoma and breast cancer cells are dependent on PHGDH for their survival, so developing PHGDH targeting inhibitor is receiving more concern for cancer diagnosis and therapeutic strategy. Inspired by this, we designed and developed fluorescence based chemical probes (DNS-pE2) with trun-on properties for live cell imaging of endogenous PHGDH. In addition to this we demonstrated its in situ engagement by using SILAC-based quantitative chemical proteomics. Our studies showed that DNS-pE2 is highly selective to endogenous PHGDH and we presume cysteins in active sites of PHGDH might enable its direct covalent modification for DNS-pE2 labeling.

 

Suna In
Code / Date
YS-2 / March 30 (Fri) 08:06-08:12
Speaker
Suna In   CV
Affiliation
KAIST
Title
Identification and characterization of eEF1BδL as a substrate of RNF20/40
Abstract

In mammalian cells, the RING-finger type E3 ubiquitin ligase RNF20/40 catalyzes mono-ubiquitylation of histone H2B that has been implicated in transcriptional regulation. RNF20 and RNF40 knockdown studies have shown defects in various cellular processes, suggesting presence of additional ubiquitylation target proteins of RNF20/40. In order to find novel non-histone target proteins of RNF20/40, we performed co-immunoprecipitation using FLAG-RNF20 cell line and subsequent mass spectrometry analysis identified eEF1BδL, the isoform 2 of elongation factor 1-delta, as an RNF20/40-interacting protein. We confirmed direct protein interaction between RNF20/40 and eEF1BδL and also found that eEF1BδL can be ubiquitylated by RNF20/40. In addition, we further showed that eEF1BδL ubiquitylation enhances expression of heat shock responsive genes. Our study demonstrates that RNF20/40 regulates gene expression through ubiquitylation of transcription factor in addition to histone H2B.

 

Nari Seo
Code / Date
YS-3 / March 30 (Fri) 08:12-08:18
Speaker
Nari Seo   CV
Affiliation
Chungnam National University
Title
Quantitation of Serum N-Glycan Isomers for Bechet Disease Monitoring
Abstract

Protein glycosylation is widely recognized as the powerful resource of biomarkers to screening disease states. Indeed, aberrant glycosylation is a well-known event in autoimmune disease and cancer. Bechet disease (BD), a typical immune disease, is a vasculitis disorder characterized by oral aphthous ulcers, genital ulcers, and uveitis. Although many diagnostic criteria of BD have been proposed, it is still challenging to establish reliable diagnosis method. Here, we developed glycomics-based novel approach for BD monitoring. Sera of Bechet patients (n=100) and health controls (n=100) were obtained from SMC (Samsung Medical Center) in Korea. Each sample was enzymatically treated with PNGase F to release N-glycans. Native N-glycans were selectively enriched by PGC-SPE prior to MS analysis. While global profiling of serum N-glycome has already revealed potential biomarkers for various types of cancer, quantitation of the aberrant glycosylation is desirable in order to improve the specificity of the diagnosis. However, the quantitation of serum glycans is analytically challenging due to the complexity caused by glycan isomers and dynamic range of glycan concentration. In this study we have developed new technology to quantify isomer-specific glycan using PGC based MRM-MS. In particular, it has been known that sialylated glycans which are highly present in immunoglobulin-related proteins are closely related to inflammation and diseases including cancers although several hundreds of glycans are present in human serum, Therefore, we decided to target sialylated glycans. Briefly, the MS was operated in positive mode using unit resolution and MRM transitions, b-ions (m/z 204.1, m/z 366.1 ([HexNAc]+, [Hex+HexNAc]+), were further selected to obtain maximum sensitivity. Interestingly, we found that BD patients contained significant quantity of mono/di-sialylated bi-antennary N-glycans consisting of Hex5 HexNAc4 NeuAc1-2 compared with healthy control group. Expression level in patient cohort were 5 to 10-folds higher than those in control group showing complete separation. Four of among eleven bi-antennary acid glycan isomers showed high diagnostic efficacy having AUC of ROC curve over 0.98. When the sensitivity was fixed to 90%, corresponding specificities were over 92% to 96%. Interestingly, these marker glycans exhibits unique correlations with biological characteristics of cohorts. Two isomers of Hex5 HexNAc4 NeuAc1 and two isomers of Hex5 HexNAc4 NeuAc2 could completely distinguish male and female groups in healthy control cohort. Glycan expression level in female groups were 6 to 10 times higher than that of male groups resulting in 1.0 AUC in ROC curve. These results could be demonstrated that the glycosylation reflected biological characteristics. Furthermore, this novel approach that targeting isomer-specific glycan will provide significant insight into the glycobiological aspects of the disease process.

 

Jinwoo Jung
Code / Date
YS-4 / March 30 (Fri) 08:18-08:24
Speaker
Jinwoo Jung   CV
Affiliation
Seoul National University
Title
PRM and SRM methods to identify prognostic biomarkers of Tocilizumab
Abstract

Rheumatoid arthritis (RA) is one of the most common chronic and systemic autoimmune diseases that cause inflammation of the tissue around the joints. Interleukin-6 (IL-6) plays a vital role in activation of local synovial leukocyte production and induction of chronic inflammation. IL-6, therefore, is an attractive therapeutic target for RA and its humanized anti-IL-6 receptor antibody,Tocilizumab (TCZ), has shown to be very effective in the treatment of patients with RA. Alothough TCZ has proven to be efficacious in patients who did not respond to other RA therapeutics, some patiens show a partial respond or resistant to the therapeutic agent. In this study, we used a proteomic approach to discover prognostic biomarkers for TCZ response from serum samples of TCZ responder and non-resoponder groups of RA patients. Potential biomarker candidates identified from serum protein profile data and public database were verified by both multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM) approaches. TCZ response predicton biomarkers candidates were further compared with prognostic biomarkers of anti-tumor necrosis facter alpha (TNF-) to discover clinical relations between the two biologics, which could complement and reinforce a beneficial therapy for RA paitens at the end.

 

RAMIN SEO
Code / Date
YS-5 / March 30 (Fri) 08:24-08:30
Speaker
RAMIN SEO   CV
Affiliation
Korea Advanced Institute of Science and Technology
Title
Establishment of 3-D Cell Culture Model for Liver Cancer
Abstract

3-D cell culture models are considered to be superior over the conventional 2-D cell culture models in recapitulating in vivo cellular features like cell-cell and cell-matrix interactions. As a model 3-D cell culture platform for liver cancer, we generated liver cancer cell spheroids using a hanging drop method. Urea and cholesterol assays are implemented to measure whether our model is mimicking native liver function. Expression level of key liver-specific enzymes, MRP2 and CYP2E1, have been examined by immunohistochemistry. Furthermore, comparative proteomic analysis by mass spectrometry has been carried out to probe the differences in protein expression profiles between the 2-D and 3-D cell culture models using the SILAC approach. The KEGG analysis and Ingenuity Pathway Analysis have indicated that the cells in the 3-D culture model displayed increased metabolism, glycolysis, and oxidoreductase activity compared to the cells in the 2-D culture model. In contrast, the expression level of proteins involved in cell cycle, apoptosis, and nucleic binding activity decreased in the 3-D model. In conclusion, our results demonstrated the efficacy of our 3-D spheroid culture platform for reproducing liver-specific cellular functions and microenvironment in cell culture system.

 

Kwang Hoe Kim
Code / Date
YS-6 / March 30 (Fri) 08:30-08:36
Speaker
Kwang Hoe Kim   CV
Affiliation
Korea Basic Science Institute
Title
Development of Fucosylated Glycopeptides in Alpha-fetoprotein Immunoprecipitated from Serum as a Biomarker Candidate for Hepatocellular Carcinoma by Parallel Reaction Monitoring
Abstract

Fucosylation is one of the most important glycosylation in the progression of hepatocellular carcinoma (HCC). Recently, fucosylated fraction of alpha-fetoprotein (AFP) has been developed as a serological marker for HCC. In this study, we introduced analytical methods for detecting glycopeptides of AFP using parallel reaction monitoring (PRM) mass spectrometry (MS) coupled with immunoprecipitation. The purpose of this study is to determine fucosylated N-glycopeptides from AFP associated with HCC using human serum. First, desialylation of target glycopeptides after immunoprecipitation of AFP was used to improve MS detection limit from sub µL serum. Second, PRM was used to obtain high resolution MS/MS spectra for identifying and quantifying target glycopeptides using hybrid quadrupole time-of-flight (Q-TOF) MS. Finally, we compared sera from HCC with liver cirrhosis using relative percentage of fucosylated glycopeptide in AFP (AFP-fuc%). AFP-fuc% showed an area under the ROC curve (AUC = 0.949, p value < 0.0001) to discriminate between HCC and cirrhosis patients. These results suggest the potential of our methods for detecting fusosylated glycopeptides of AFP from human serum. Also, we demonstrate the possibility of AFP-fuc% as a promising parameter for HCC.

 

Hee-Sung Ahn
Code / Date
YS-7 / March 30 (Fri) 08:36-08:42
Speaker
Hee-Sung Ahn   CV
Affiliation
UST
Title
SEPROGADIC – serum protein-based gastric cancer prediction models for prognosis and selection of proper adjuvant therapy.
Abstract

Gastric cancer patients usually receive surgical treatment. Postoperative medical options based on prognostic prediction models are mostly related with anticancer adjuvant therapies (AT) and would provide a patient-specific treatment to extend the life span of patients. Novel protein markers of prognosis and blood tests using them will help to solve unmet clinical needs. For this, we designed a retrospective study in which serum samples were collected from the patients at a 4-week recovery period after D2 lymph node dissection and their protein marker candidates were detected by multiple reaction monitoring mass spectrometry and quantified to validate useful biomarkers for applying postoperative clinical practices. We then developed the SEPROGADIC (SErum PROtein-based GAstric cancer preDICtor) prognostic model that clearly classified the patients with good or bad prognosis at each of the clinical stage and identified a patient group who would have benefited from CCRT (combined chemoradiation therapy) rather than CTX (chemotherapy). Our study demonstrates that serum proteins of cancer patients could be a mirror that reflects the disease-related state at the molecular level and plays an important bridge in medical decision-making.

 

Seunghyup Jeong
Code / Date
YS-8 / March 30 (Fri) 08:42-08:48
Speaker
Seunghyup Jeong   CV
Affiliation
GRAST, Chungnam National University
Title
Aberrant glycosylation is associated with gastric cancer and precancerous diseases
Abstract

Gastric cancer is one of the most commonly occurring malignancy and leading cause of cancer death in Korea. Classically, CEA, CA19-9, and CA 72-4 have been used for non-invasive gastric cancer screening. However, current protein markers are not gastric cancer specific, and also have low specificity and sensitivity. Glycosylation is the most common post-translational modification and plays an important role in various biological processes. Glycans in blood are secreted from cells in the form of glycoprotein and these glycans are changed depending on health condition and diseases including cancers. Aberrant glycosylation is the mainly observed in various types of cancer cases (e.g. cancer cells and serum of cancer patients). Glycan profiling of whole serum is already developed and widely used in cancer biomarker study. However, targeted glycoproteomic approach is needed in clinical field for better sensitivity and specificity. In previous study, we targeted serum haptoglobin and explored the glycan alterations between gastric cancer patient and healthy control. Based on these glycan signatures, we studied aberrant glycosylation on intact glycoprotein haptoglobin without enzymatic release and enrichment process for rapid, high-throughput clinical screening. Like glycan level, intact haptoglobin represent significant differences between cancer patients (n = 44) and healthy controls (n = 44) and we found high grade marker (AUC = 0.81 to 0.93). Based on previous study, here, we have applied to samples not only gastric cancer patients but also various kinds of precancerous gastric diseases (including atrophic gastritis, gastric ulcer, and gastric dysplasia) for gastric disease biomarker discovery. Samples were analyzed by UHPLC-Q-TOF MS with only denaturation process for sample preparation. Standards shows high reproducibility (n = 27, CV < 10%) while clinical samples represent high individual variation (CV > 26%) even same sample groups. Despite individual variation, each groups clustered same kinds of samples and shows significant differences between control and disease groups. This study can provide the information to distinguish and diagnose gastric cancer and other precancerous gastric diseases.

 

Wooyoung Eric Jang
Code / Date
YS-9 / March 30 (Fri) 08:48-08:54
Speaker
Wooyoung Eric Jang   CV
Affiliation
Kyung Hee University
Title
Application of LC-MS/MS to identify biomarkers for autism spectrum disorder using Cntnap2 KO mouse
Abstract

Autism spectrum disorder (ASD) represents a disorder related to socio-psychological and neurodevelopmental problems. [2] In the United States of America alone, 1 out of 64 children suffers from ASD. [1]ASD is an early onset developmental disorder that affects the patients in life-long impact on behavior and social functions. Fortunately, autistic behaviors if treated in early stages the patients can live their life with normal social functions and behaviors. [4] However, there is no molecular early diagnostic marker available. In 2006 it was noted that Cntnap2 is a recessive mutation that is a syndromic form of ASD called cortical dysplasia-focal epilepsy syndrome. This mutation cause seizures, language regression, intellectual disability, hyperactivity and most of the patients have a degree of ASD. [3] Using Cntnap2 KO mice and utilizing proteomics method, we aim to achieve in-depth profiling of tissue proteomes of the autistic animal model. The Cntnap2 KO mouse brain was dissected to isolate hippocampus, mPFC, and cerebellum. For quantification in discovery experiments, the isobaric TMT reagents (Tandem Mass Tag) were used to label peptides prepared from the mouse brain tissues. The raw mass spectrometry data were analyzed using the MaxQuant software. Quantitative protein intensities were filtered using fold change (log) greater than 0.5 and p-value less than 0.05. These proteins were used to carry out bioinformatics Gene Ontology analysis to find affected biological pathways by the mutation. Top 20 up-regulated proteins and top 20 down-regulated proteins satisfying p-value less than 0.05 were evaluated. Using volcano plot, significant proteins were identified and these proteins were further studied. So far we identified that protein Tsc1 is down-regulated and this protein is part of the mTOR biological pathway which is related to Autism. Cacna1h is up-regulated and this protein is related to calcium channel biological pathway which is correlated with ASD. Gene Set Enrichment Analysis (GSEA) was implemented between the KO/WT groups. The mPFC, hippocampus and cerebellum data will be compared for biomarker candidates. Using biomarker candidates, MRM qualification experiment will be proceeded using Human plasma samples. The hope for the future is that by using the non-invasive method, clinicians will be able to diagnose young patients with ASD.

 

Jong Hyuk Yoon
Code / Date
YS-10 / March 30 (Fri) 08:54-09:00
Speaker
Jong Hyuk Yoon   CV
Affiliation
Korea Brain Research Institute
Title
Multidimensional proteomic analysis for understanding Alzheimer's disease
Abstract

Neuronal degeneration interferes the function of neural circuits damaged by neuronal plaques. Diagnostics of Alzheimer’s disease is still difficult by using clinically available methods such as clinical exams and amyloid plaque imaging. Nano-sized extracellular vesicular entities believed to travel the blood brain barrier carrying neuronal proteins and the diseased brain condition may change the profile of vesicular contents released into the blood vessels from the brain. Extracellular vesicles (EVs) have been focused as fascinating targets since they contains signaling proteins, lipids and nucleic acids but there is less report about blood EVs because of difficulty in analysis for contents profiling. To target and reveal the vesicular contents of blood vesicles released in Alzheimer’s plaque containing brain, we combined multidimensional proteomics and informatics tools for profiling of blood EVs in the TG6799 Alzheimer’s mouse model. We identified TG6799 specifically changed blood EV proteins according to reliable label-free quantitative proteomic analysis. Based on subsequent informatics and biochemical experiments, we selected the AD protein biomarker candidates. We found AD-B1 (AD biomarker #1) was transported from neurons to EVs by -amyloid dependent deSUMOylation. AD-B2 (AD biomarker #2) was also affected by -amyloid treatment in its secretion and enzymatic activity. We finally validated those AD protein biomarker candidates using human specimens. Furthermore we identified lipids that are specifically changes in human AD blood EVs using new method for lipid MALDI-TOF analysis. This study provides new molecular signatures for AD diagnostics and molecular characterization of AD pathology.

 

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