Speaker : Chang Ho Sohn, Ph.D. NBC COLLOQUIUM Part I. Novel Chemical Labeling Approach in Mass Spectrometry (MS)-Based Proteomics Part II. Toward Understanding of Single Cell Signaling Dynamics by Time-Lapse Fluorescence Microscopy and Optogenetics Speaker : Chang Ho Sohn, Ph.D. California Institute of Technology Part I> Novel Chemical Labeling Approach in Mass Spectrometry (MS)-Based Proteomics Quantifying protein expression level is a key task in MS-based proteomics. Caltech isobaric tags (CITs) for protein quantification were developed and validated using various model samples. A newly discovered low-energy gas-phase fragmentation pathway, a nucleophilic substitution of the N3 in the 1,2,3-triazole ring generated by copper-catalyzed  azide-alkyne cycloaddition (CuAAC) inspired us to create CITs. This selective cleavage is applied to the formation of the reporter ions to quantify protein expression level in cells. Clickable cross-linkers (CXLs) were developed for elucidation of three-dimensional protein structures and protein-protein interactions (PPIs). In CXLs, cross-linking reactions are separated from the conjugation of affinity tags, avoiding steric hindrance. Cross-linked peptides are enriched from the complex mixture of yeast lysate and cross-linked ubiquitin digests using avidin affinity chromatography, showing high sensitivity of the CXL-based analysis. The low-energy pathway used for CIT reagents is also adopted to produce the reporter ion, filtering MS/MS scans of cross-linked peptides from those of unmodified peptides. Part II> Toward Understanding of Single Cell Signaling Dynamics by Time-Lapse Fluorescence Microscopy and Optogenetics Recent advances in fluorescence microscopic techniques allow us to investigate single cell gene expression/functional dynamics. Especially pulsatile behavior of transcription factor localization was observed in many species (sigma factor in E. coli, Crz1 in yeast, NF-kB, ERK1 and p53 in mammalian cells), indicating their regulating roles in cell signaling pathways. Localization dynamics of a transcription factor, Mig1 in S. cerevisiae which governs lots of the downstream genes associated with glucose repression was investigated using time-lapse microscope. The pulsatile bursting localization dynamics was revealed by Mig1-mcherry time-lapse imaging under glucose stress. To understand this behavior, phosphorylation catalytic interactions with upstream kinase (Snf1) and phosphatase (Glc7-Reg1) were induced by light-controlled binding of optogenetic tool kits-PhyB-PIF6 protein fusions. Implications on the mechanism of the bursting cell signals will be discussed. 4:00 PM on Nov. 28(THU) Engineering Building 1, E104 Prof. Oh-Hoon Kwon(5204, ohkwon@unist.ac.kr) NBC Office (2642, aekang@unist.ac.kr)