Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

The use of unique peptide-tags for sensitive detection of yeast proteome   (#620)

Keiji Kito 1
  1. Meiji University, Kawasaki, KANAGAWA, Japan

Technical advances in mass spectrometry (MS)-based proteomic analysis have enabled proteome-wide analysis with high coverage and sensitivity. Detection of individual proteins generally relies on peptide identification via tandem mass analysis. Therefore, successful detection of each protein depends on the presence of proteolytic peptide sequences that are suited for detection and identification by MS analysis. However, a subset of proteins, especially of small molecular weight or consisting of biased sequence, contain few peptide sequences that can be detected with high sensitivity and generate good quality of tandem mass spectra. In addition, extreme complexity in protein sample makes it difficult to detect low-abundance proteins with high dynamic range.

To overcome these problems, we have developed a strategy using a genetically modified yeast strain in which each protein is tagged with unique peptide-tag suited for MS detection and identification. Only peptide-tags are purified and subjected to MS analysis, leading to a reduction in sample complexity and allowing for sensitive detection even in the case of low-abundance proteins. A single yeast strain in which unique peptide-tags, which were selected from peptide library generated from DNA template produced by degenerate PCR and chemically synthesized, were fused to multiple proteins was successfully constructed with high efficiency by the use of CRISPR/Cas9 system. We present a proof of concept of this strategy using such a yeast strain best-optimized for proteomic analysis.