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Heeyoun Hwang
Code / Date
YS-1 / March 29 (Tue) 14:30-14:40
Speaker
Heeyoun Hwang
Affiliation
Korea Basic Science Institute
Title
Integrated Proteomic Pipeline using Multiple Search Engines for Chromosome-based Proteomic Study from Human Hippocampal Tissue
Abstract

The goal of the chromosome-based proteomic study is to fully provide proteomic information from each human chromosome, including missing protein as well as novel proteoforms which are expressed from non-coding genomic regions, Alternative Splicing Variants (ASVs), and Single Amino-Acid Variants (SAAVs).
For the chromosome-based proteomic study, we developed a workflow including Integrated Proteomic Pipeline (IPP) which consists of three different search engines as well as statistical evaluation tools combined with a homemade program for the normalization of three scores from different search engines.
From 144 LC/MS/MS raw files from human hippocampal tissues of control, epilepsy and Alzheimer Disease (AD), we identified 5,752 proteins including 503 ASVs and three missing proteins with a less than 1% False Discovery Rate (FDR) at the protein level. Additionally, four Translated long non-coding RNA Variants (TRVs) and four novel ASVs were identified against the customized databases of GENCODE. The target peptides of the missing proteins and variants were validated by tandem MS fragmentation pattern from their corresponding synthetic peptides. Last, a total of 128 SAAVs paired with their wild-type peptides were identified with FDR < 1% at the peptide level using a customized database from neXtProt including non-synonymous single nucleotide polymorphism (nsSNP) information.
Among these results, several novel variants related in neuro-degenerative disease were identified using the workflow that could be applicable to C-HPP studies.

 

Eunjin Yu
Code / Date
YS-2 / March 29 (Tue) 14:40-14:50
Speaker
Eunjin Yu
Affiliation
KAIST
Title
Interactome Analysis of Insect Olfactory Receptor Co-Receptor From the Adult Drosophila
Abstract

Insects distinguish food, poison, mate, and enemy mainly by odors; studies of insect olfaction may answer questions about their behavior, and in turn, will benefit human welfare by helping build up a better symbiotic relationship between the two species. Interestingly, insect olfaction is molecularly distinct from vertebrate and nematode olfaction such that the insect odorant receptors (ORs) comprise a unique class of chemoreceptor. The functional characteristics of ORs, as ligand-gated cation channels, have been extensively studied at the neuronal level, whereas their molecular mechanisms are still obscure. To investigate the molecular mechanisms underlying OR functions, we have focused on the fact that different ORs interact with a single obligatory binding partner Orco, an insect-specific co-receptor. Initially mischaracterized as one of the ORs, Orco, like ORs, is a membrane protein spanning the plasma membrane seven times with its N-terminus in the cell. Unlike ORs, however, Orco does not respond to natural odors and its homologs from different insect species share a high sequence identity. We first sought to identify Orco interactome by immuno-precipitation followed by mass spectrometry (IP-MS). To this end, we have developed a methodology that enables tissue-specific extraction of intact protein complexes from adult Drosophila, a rarely achieved feat due to technical limitations. In our methodology, the antennal tissues, where most of Orco is expressed, are enriched with a custom-made sieve. After milling, which helps break up the chitinous exoskeleton, the protein complexes in the antenna-enriched tissues are extracted using a mass spec-compatible detergent lauryl maltoside. Using this methodology combined with IP-MS, we identified novel Orco binding partners that are currently undergoing orthogonal confirmations. We expect our novel protein-level screening methodology to provide important clues to insect olfactory mechanisms and to be broadly applicable to tissue-specific fly interactome analysis.

 

Jeonghun Yeom
Code / Date
YS-3 / March 29 (Tue) 14:50-15:00
Speaker
Jeonghun Yeom
Affiliation
KIST-UST
Title
Getting protein-level evidence of genomic variant in stomach cancer cells by a proteogenomic approach
Abstract

Variations in the coding sequence of genes causing amino acid codon alterations may lead to protein misfolding, polarity shift, improper phosphorylation and other functional consequences. Since expressions of such variants are often suppressed by cellular quality control systems, it is required to confirm their protein-level expression. The goal of our study was to identify genomic variants at the protein level, and to monitor the expression of such variants from DNA to protein. Toward this, we have developed a proteogenomic strategy getting protein-level evidence of genomic variants, which we call sequential targeted LC-MS/MS based on prediction of peptide pI and RT (STaLPIR). To support our theory, we analyzed three gastric cancer cell lines by shotgun genomics and proteomics. By comparing the MS data to exome-sequencing data, we chose a set of 296 nonsynonymous variations and put onto STaLPIR. Finally, we were able to confirm the expression of 147 variants. We found different expression pattern of variants according to allele type (reference (AA), hetero (AB) and variant (BB)) during expression of DNA to protein. Integrated analysis of DNA, mRNA and protein suggests that protein expression level of the nonsynonymous variant is regulated either before or after translation, according to influence of the variant on protein function. In conclusion, our data provides an excellent approach getting direct evidence for the expression of variant protein forms from genome sequence data.

 

Youngsuk Seo
Code / Date
YS-4 / March 29 (Tue) 15:00-15:10
Speaker
Youngsuk Seo
Affiliation
Chungnam National University
Title
Site-specific glycosylation analysis of biotherapeutics with micro-heterogeneity and macro-heterogeneity
Abstract

Glycosyl modification of therapeutic glycoproteins is significantly associated with drug efficacy, immunogenicity, and stability. Thus, comprehensive glycomic analyses including the determination of sites and glycan heterogeneities are required for biotherapeutic quality assurance/quality control and assessment. Herein, we designed new avenue to explore site-specific glycoform using a signature glycopeptide which is an unique molecule to provide the information of both glycosylation site and heterogeneity in a biopharmaceutical glycoprotein. Our strategy involves digestion of a glycoprotein with (multi)-specific protease to produce signature glycopeptides, glycopeptide filtration to separate N- & O-glycopeptides, and enrichment for signature biomolecules using SPE followed by nano LC-MS and LC-MS/MS. To evaluate our analytical platform, we examined 2nd EPO which is a representative biotherapeutic glycoprotein possessing multiple N- & O-glycosylation sites. EPO was initially treated with trypsin followed by filtration using MWCO spin column to separate N-glycopeptides from O-glycopetides. Tryptic N-glycopeptides were further digested with non-specific proteases to produce a signature glycopeptide at each glycosylation site. We could obtain signature N-glycopeptides consisting of a glycan moiety and a short peptide tag. Glycopeptides were putatively assigned with accurate masses and their structures were further confirmed by tandem MS. Using signature glycopeptides we successfully mapped EPO glycosylation showing glycan heterogeneity (micro-heterogeneity) and site occupancy with different degree (abundance) of glycosylation (macro-heterogeneity). Indeed, we found that three N-glycosylation sites (Asn 38, 83, 88) are heavily glycosylated while others (Asn 24, 30) have less glycans due to steric hindrance. The combined information using qualitative and quantitative profiling of signature glycopeptides enables comprehensive characterization of site-specific mapping with micro-heterogeneity and macro-heterogeneity.

 

Min Jueng Kang
Code / Date
YS-5 / March 29 (Tue) 15:10-15:20
Speaker
Min Jueng Kang
Affiliation
Seoul National University
Title
PRM-MS/MS for identification of rheumatoid arthritis specific citrullinated peptides in synovial fluid
Abstract

The protein/peptide citrullination is a post-translational modification and the citrullinated peptide epitopes are recognized by anti-citrullinated protein/peptide antibody (ACPA), which is a major diagnostic or prognostic marker for rheumatoid arthritis (RA). Together with ACPA, specific citrullinated endogenous protein/peptide autoantigens in the major inflammatory lesions of RA (e.g, synovial fluid) may provide valuable information for the development of early diagnosis and therapeutic intervention. To identify citrullinated peptide in synovial fluids, we pooled synovial fluids obtained from RA (n=4) and osteoarthritis (OA) (n=4) patients separately. Peptides in each pooled synovial fluid were extracted using centrifugal filtration and solid phase extraction, and then analyzed by a Q-Exactive mass spectrometer. ScaffoldPTM (v.2.1.3) was used to validate peptide identification and citrullination site mapping. Elevated levels of RA-specific citrullinated peptides are validated by PRM-MS/MS analysis with synovial fluids from RA (n=30), OA (n=15), and ankylosing spondylitis (AS) (n=5).
We have identified total 1094 endogenous peptides originated from 120 proteins using LC-MS peptide profiling experiment with pooled RA and osteoarthritis (OA) synovial fluids, separately. Among them 121 peptides derived from 9 proteins (FGA, FGB, SAA1, HMGN2, PTMA, SRGN, COL5A3, PRG4, and APOBR) were found to be citrullinated. The majority of citrullinated peptides were detected only in RA synovial fluid where some identified FGA citrullinated peptides are previously known citrullination sites of FGA, which validate our experiment. We also identified an RA unique citullinated peptide, which has not been reported in the past. To verify RA-specific citrullinated peptides, multiplexed PRM-MS/MS analysis of selected citullinated peptides with synovial fluids from RA (n=30), OA (n=15), and ankylosing spondylitis (AS) (n=5) patients was conducted. As a results, 14 citrullinated peptides from four proteins (FGA, FGB, SAA1 and PRG4) were significantly elevated in the RA compare to the OA and AS synovial fluids (3.52 < Log2 Fold-Change < 8.65, p-value < 0.03). The eventual results of independent cohort studies confirm the possible pathophysiological link between citullinated peptide markers and RA, which will provide their diagnostic or therapeutic values for RA.

 

Ba Reun Kim
Code / Date
YS-6 / March 29 (Tue) 15:20-15:30
Speaker
Ba Reun Kim
Affiliation
Pusan National University
Title
Novel Peptides based the First FAS I Domain of Periostin for Angiogenic therapy in Murine dermal wound model.
Abstract

Angiogenic peptide drugs have a potential of clinical application in treating severe ischemic diseases. Periostin, an extracellular matrix protein expressed in injured tissues, has been reported to promote angiogenesis and tissue repair. In previous study, we showed that the first FAS-1 domain in periostin is responsible for periostin-induced migration of endothelial colony forming cells (ECFCs) and demonstrated that periostin could be a therapeutic agent for the treatment of peripheral artery occlusive disease by promoting homing of ECFCs. In this study, we further characterized the first FAS-1 domain of periostin to identify a minimum region responsible for promoting ECFCs migration and angiogenic activity. A peptide from 142th amino acid to 151th amino acid in the first FAS-1 domain of periostin was critical for increasing chemotatic migration, adhesion, proliferation and tubulogenesis of human ECFCs in vitro. Chemotatic migration of ECFCs induced by the periostin peptide was abrogated by blocking antibody against β5 integrin. In addition, the periostin peptide activated AKT/ERK signaling pathway. Moreover, in the skin wound, treatment of the periostin peptide accelerated wound closure compared with HBSS-control group. These results suggest that the periostin peptide has a potential as a drug candidate for enhancing angiogenesis and wound closure.

 

Hyuck Jun Mok
Code / Date
YS-7 / March 29 (Tue) 15:30-15:40
Speaker
Hyuck Jun Mok
Affiliation
Kyung Hee University
Title
A rapid and sensitive profiling of lysophosphatidic acid using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) after chemical derivatization in ovarian cancer stem cells
Abstract

Lysophosphatidic acid (LPA), a simple phospholipid, functions as a signaling molecule in cell proliferation, survival, adhesion, migration, and differentiation. Cancer stem cells (CSCs) are known to secrete autocrine factors that promote a neighboring environment favorable for their self-renewal and maintenance. In this study, investigated the factors secreted from ovarian CSCs which stimulates migration and proliferation of CSCs. Elevated LPA in ascites of ovarian cancer patients and its involvement in growth and invasion of ovarian cancer cells have been reported that ovarian cancer shows high mortality due to development of resistance to chemotherapy and relapse. As LPA biology is booming, the development of method for identification and quantification of LPA is needed. However, the accurate analysis of low-abundance acidic PLs such as LPA is hindered by peak tailing and low detection sensitivity caused by metal affinity of phosphate group. We developed a method for identification and quantification of LPA based on chemical derivatization and liquid chromatography-mass spectrometry (LC-MS). Of various derivatization methods, methylation is one of effective method to derivatize the phosphate group. In the present study, we applied trimethylsilyldiazomethane (TMSD) derivatization to improve ionization efficiencies in the profiling of LPAs. Multiple reaction monitoring (MRM) was used for the selective quantification and detail identification of methylated LPA. Our novel profiling method revealed that LPA, especially LPA (18:1) was significantly elevated in CSC culture media compared with non-CSC culture media.