Puromycin-Modified Silica Microsphere-Based Nascent Proteomics Method for Rapid and Deep Nascent Proteome Profile

Area of Science:

• Proteomics
• Molecular Biology
• Biochemistry

Background:

• Nascent proteome analysis reveals gene regulation at the translational level.
• Current puromycin capture methods struggle with low-abundance nascent proteins, limiting deep proteome profiling.

Purpose of the Study:

• To develop an efficient method for capturing and analyzing nascent proteins.
• To overcome the limitations of low abundance in nascent protein identification.

Main Methods:

• Synthesis of puromycin-modified silica microspheres (PMSs) for dispersive solid-phase microextraction.
• Establishment and optimization of the PMS-based nascent proteomics (PMSNP) method.
• Application of PMSNP in HeLa cells and mouse brains, including a biological stress model.

Main Results:

• High modification efficiency (91.8%) of puromycin groups on silica microspheres via click reaction.
• Rapid identification of over 3500 (HeLa cells) and 3900 (mouse brains) nascent proteins within 13.5 hours.
• Successful application in a lipopolysaccharide-stimulated HeLa cell model, revealing unique nascent proteome characteristics.

Conclusions:

• The PMSNP method provides efficient capture and analysis of nascent proteins, including low-abundance and secreted ones.
• Demonstrates high sensitivity and immediacy for studying translational regulation and biological stress responses.
• Offers a superior approach for deep profiling of the nascent proteome.