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  • SYM-1Proteomics for overcoming metabolic diseases SYM-1 View
  • SYM-2Proteomics for understanding brain functions SYM-2 View
  • SYM-3New innovations in proteomics SYM-3 View
  • SYM-4Proteomics in multi-disciplinary approaches SYM-4 View
  • SYM-5Proteomics for precision medicine SYM-5 View
  • SYM-6Proteomics for stem cell medicine SYM-6 View

SYM-3 : New innovations in proteomics

Organizer : Jun-Seok Lee (Korea Institute of Science and Technology, Korea)

The purpose of “New Innovations in Proteomics” session is to provide introductory lectures of novel proteomics tool development and their application in disease biomarker discovery and mechanism study.



Gavin E. Reid
Code / Date
SYM 3-1 / March 28(Mon) 16:50-17:04
Speaker
Gavin E. Reid   CV
Affiliation
The University of Melbourne, Australia
Title
A Quantitative Mass Spectrometry-Based Assay for the Clinical Analysis of Parathyroid Hormone and Its Heterogeneous Oxidized and Truncated Post Translational Modifications
Abstract

Parathyroid hormone (PTH) plays a critical role in the regulation of circulating blood calcium levels, and serves as a biomarker of secondary hyperparathyroidism associated with the diagnosis of disorders such as vitamin D deficiency or chronic kidney disease (CKD), and for monitoring the effectiveness of treatment e.g., hemodialysis. Immuno-chemiluminescent assays for full length (i.e., 1-84) PTH have historically been problematic due to variable standardization, cross reactivity with truncated PTH fragments (e.g., 7-84), and variable correlations with biological parameters. A recent report that PTH may be oxidized in vivo further complicates these issues, since oxidized PTH is biologically inactive.
To overcome these challenges, I will describe in this presentation the development and application of an alternate strategy for analysis of a series of clinical hemodialysis patient plasma samples, involving immunoaffinity enrichment, liquid chromatography–tandem mass spectrometry (LC-MS/MS), and a novel dual isotope-labeled internal standard quantitation approach to (i) quantitatively determine the nature of circulating full length and truncated PTH and their individual oxidized isoforms, (ii) differentiate between in vivo and ex vivo oxidative modifications, and (iii) evaluate the effects of PTH oxidation on current immunochemiluminescent PTH assays.

 

Erika Noro
Code / Date
SYM 3-2 / March 28(Mon) 17:04-17:18
Speaker
Erika Noro   CV
Affiliation
National Institute of Advanced Industrial Science and Technology (AIST), Japan
Title
Large-scale identification of glycoproteins carrying Lewis x and site-specific glycan alterations in Fut9 knockout mice
Abstract

The Lewis x (Le x) structure (Galβ1-4(Fucα1-3)GlcNAc-R) is a carbohydrate epitope comprising stage-specific embryonic antigen-1 (SSEA-1) and CD15, and is synthesized mainly with α1,3-fucosyltransferase 9 (Fut9) in mouse. Fut9 gene is expressed specifically in the stomach, kidney, brain, and leukocytes, suggesting that Lex has a specific function in these tissues. However, there is little information for glycoproteins carrying Lex. We identified the glycoproteins carrying Lex utilizing a newly developed glycoproteomic technology for site-specific glycome analysis, and revealed global glycan alterations associated with knockout (KO) of the Lex-synthesizing glycosyltransferase gene, Fut9.
In this study, we first profiled the N-linked glycans of kidney glycoproteins in wild-type (WT) mice by MS. The analysis revealed that most of complex-type N-glycans have core fucose and significant part of the glycans have terminal fucoses recognized by presence of multiple fucoses on single N-glycan. Since the putative terminal fucoses were lost following KO of Fut9, the terminal fucoses were concluded to be Lex. Next, glycopeptides carrying fucose(s) were captured with a fucose-binding lectin, Aleuria aurantia lectin (AAL)-affinity chromatography from WT mouse kidney, and their peptide portions were identified comprehensively by the IGOT-LC-MS method (Kaji et al., Nat Biotechnol., 2003). Among the identified glycopeptides, we assigned glycopeptides carrying multiple fucoses, i.e., Lex, by the newly developed site-specific glycan analysis method, named Glyco-RIDGE (Noro et al., J Proteome Res., 2015). The method composed of dissociation-independent assignment of glycopeptide signals and accurate mass-based prediction of N-glycome on the glycopeptides. Our analyses demonstrated that 24 out of 32 glycoproteins carry Lex-containing N-glycans in WT kidney. By comparison with the results between KO and WT mice, disappearance of the Lex motifs was confirmed in 21 of the 24 Lex carriers in the KO mice. These results indicated presence of Lex on widespread glycoproteins rather than limited specific proteins. Therefore, the number of detectable glycopeptides was lower than anticipated. However, despite the glycan variation on each glycopeptide, we could reveal 522 glycopeptide signals, 51 glycosylated sites in 24 glycoproteins in WT mice, and 591 glycopeptide signals, 70 glycosylated sites in 48 glycoproteins in KO mice. These numbers are supposed to be the largest that have been reported so far. Furthermore, we compared the glycan structures on kidney glycoproteins between WT and KO mice. In WT, a little sialylated glycans were detected; however, addition of sialic acids was increased in Fut9 KO. Because terminal fucosylation (Lex formation) competes with sialylation, abundant Lex inhibits sialylation in WT kidney. Therefore, Fut9 might play a role in keeping the kidney cell surface in a neutral state by protecting the glycan against sialylation and leading a negative charge state. This is the first report of large-scale identification of Lex-carrying glycoproteins from a native sample based on the site-specific glycome analysis.

 

Hyun-Suk Lim
Code / Date
SYM 3-3 / March 28(Mon) 17:18-17:32
Speaker
Hyun-Suk Lim   CV
Affiliation
Pohang University of Science and Technology (POSTECH), Korea
Title
Targeting Protein-Protein Interactions Using Proteomimetics
Abstract

The majority of drugs on the market today target proteins with defined small-molecule binding sites, including enzymes and receptors. However, some of the most pressing and devastating diseases involve proteins that do not possess these natural binding sites, such as those involved in protein-protein interactions associated with many cancers. Molecules capable of modulating protein-protein interactions are thus valuable research tools to uncover molecular functions of target proteins and further can be potential therapeutic candidates. However, discovering such molecules is challenging mostly due to the fairly large and flat protein interfaces involved in protein-protein interactions. Typical drug-like small molecules may not be suitable to effectively cover such extended protein contact areas. Thus, there is an urgent need for the development of different types of molecules to target protein interfaces. In order to address these fundamental problems, we have made considerable efforts to design and synthesize a novel class of synthetic molecules, called proteomimetics that can mimic protein surface structure and function. Here I will present recent development of such proteomimetics and their biological application.

 

Jung-Min Kee
Code / Date
SYM 3-4 / March 28(Mon) 17:32-17:46
Speaker
Jung-Min Kee   CV
Affiliation
Ulsan National Institute of Science and Technology (UNIST), Korea
Title
Phosphohistidine, a Dark Matter in the Phosphoproteome
Abstract

My research program focuses on the synergistic combination of synthetic organic chemistry and protein chemistry for innovative applications in biology and medicine. I will discuss our work on phosphohistidine (pHis), a labile protein post-translational modification (PTM). Despite the long-recognized importance of pHis in cell signaling and metabolism, the full scope and exact function of protein histidine phosphorylation is not clearly understood. It is mainly due to the chronic lack of adequate research tools to investigate the unstable PTM. We provided first glimpse at this “dark matter” in the phosphoproteome through the development of novel chemical tools for studying histidine phosphorylation, including stable pHis analogs, pHis-specific antibodies and their application in phosphoproteomics.

[1] Kee,J.-M.; Oslund, R. C.; Couvillon, A. D.; Muir, T. W.* Org. Lett. 2015, 17, 187-189.
[2] Oslund, R. C.†; Kee,J.-M.†; Couvillon, A. D.; Bhatia, V.; Perlman, D. H.*; Muir, T. W.* J. Am. Chem. Soc. 2014, 136, 12899–12911 (†Equalcontributions)
[3] Kee,J.-M.; Oslund, R. C.; Perlman, D. H.; Muir, T. W.* Nat. Chem. Biol., 2013, 9, 416–421.
[4] Kee,J.-M.; Muir, T. W.* ACS Chem. Biol. 2012, 7, 44-51.
[5] Kee,J.-M.; Villani, B.; Carpenter, L. R.; Muir, T. W.* J. Am. Chem. Soc. 2010, 132, 14327-14329.

 

Hyun-Woo Rhee
Code / Date
SYM 3-5 / March 28(Mon) 17:46-18:00
Speaker
Hyun-Woo Rhee   CV
Affiliation
Ulsan National Institute of Science and Technology (UNIST), Korea
Title
Spot-ID: Identification of Biotin-Labeled Sites by APEX-and pBirA for In Vivo Interactome Mapping
Abstract

Proteins are dynamically distributed in living cellsand it is important to know each protein’s spatiotemporal localization in living cells. In mylaboratory, we have recently developed anew method to identify biotin-labeled sites of APEX (engineered ascorbate peroxidase)1and pBirA (promiscuous biotin ligase)2by mass spectrometry. Our new method(or Spot-ID)clearlyshowed unbiased result of APEX-or pBirA-labeled proteome. This method is successfully employed to identify sub-mitochondrion proteome and rapamycin-induced interactome mapping of mTOR in living cell. Furthermore,these sites contain valuable informationfor determination of native structureof labeled protein in living dynamic conditionsbecause theseenzymatic protein labeling reactionsarepreferentially occurredon thesurface-exposed residues of labeled proteins. In this seminar, our recent Spot-ID results will be presented and discussed.