Development of Multiplexed Immuno-N-Terminomics to Reveal the Landscape of Proteolytic Processing in Early Embryogenesis of Drosophila melanogaster
- Sanghee ShinSanghee ShinCenter for RNA Research, Institute for Basic Science, Seoul 08826, KoreaSchool of Biological Sciences, Seoul National University, Seoul 08826, KoreaMore by Sanghee Shin
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- Ji Hye HongJi Hye HongCenter for RNA Research, Institute for Basic Science, Seoul 08826, KoreaSchool of Biological Sciences, Seoul National University, Seoul 08826, KoreaMore by Ji Hye Hong
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- Yongwoo NaYongwoo NaCenter for RNA Research, Institute for Basic Science, Seoul 08826, KoreaSchool of Biological Sciences, Seoul National University, Seoul 08826, KoreaMore by Yongwoo Na
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- Mihye LeeMihye LeeSoonchunhyang Institute of Medi-bio Science, Soonchunhyang University, Cheonan-si, Chungcheongnam-do 31151, KoreaMore by Mihye Lee
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- Wei-Jun QianWei-Jun QianIntegrative Omics, Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, United StatesMore by Wei-Jun Qian
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- V. Narry KimV. Narry KimCenter for RNA Research, Institute for Basic Science, Seoul 08826, KoreaSchool of Biological Sciences, Seoul National University, Seoul 08826, KoreaMore by V. Narry Kim
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- Jong-Seo Kim*Jong-Seo Kim*Email: [email protected]Center for RNA Research, Institute for Basic Science, Seoul 08826, KoreaSchool of Biological Sciences, Seoul National University, Seoul 08826, KoreaMore by Jong-Seo Kim
Abstract
Protein expression levels are regulated through both translation and degradation mechanisms. Levels of degradation intermediates, that is, partially degraded proteins, cannot be distinguished from those of intact proteins by global proteomics analysis, which quantify total protein abundance levels. This study aimed to develop a tool for assessing the aspects of degradation regulation via proteolytic processing through a new multiplexed N-terminomics method involving selective isobaric labeling of protein N-termini and immunoaffinity capture of the labeled N-terminal peptides. Our method allows for not only identification of proteolytic cleavage sites, but also highly multiplexed quantification of proteolytic processing. We profiled a number of potential cleavage sites by signal peptidase and provided experimental confirmation of predicted cleavage sites of signal peptide. Furthermore, the present method uniquely represents the landscape of proteomic proteolytic processing rate during early embryogenesis in Drosophila melanogaster, revealing the underlying mechanism of stringent decay regulation of zygotically expressed proteins during early stages of embryogenesis.
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