September 2, 2020
Journal Article

Development of Multiplexed Immuno-N-Terminomics to Reveal the Landscape of Proteolytic Processing in Early Embryogenesis of Drosophila melanogaster

Weijun Qian
Sanghee Shin
Ji Hong
Yongwoo Na
Mihye Lee
Jong-Seo Kim
V Kim

Abstract

Protein expression levels are regulated through both translation and degradation mechanisms. Levels of degradation intermediates, i.e., 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 or 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.

Revised: September 2, 2020 | Published: April 7, 2020

Citation

Shin S., J. Hong, Y. Na, M. Lee, W. Qian, V.N. Kim, and J. Kim. 2020. "Development of Multiplexed Immuno-N-Terminomics to Reveal the Landscape of Proteolytic Processing in Early Embryogenesis of Drosophila melanogaster." Analytical Chemistry 92, no. 7:4926-4934. PNNL-SA-149241. doi:10.1021/acs.analchem.9b05035