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

Nascent
proteome is crucial in directly revealing how the expression
of a gene is regulated on a translation level. In the nascent protein
identification, puromycin capture is one of the pivotal methods, but
it is still facing the challenge in the deep profiling of nascent
proteomes due to the low abundance of most nascent proteins. Here,
we describe the synthesis of puromycin-modified silica microspheres
(PMSs) as the sorbent of dispersive solid-phase microextraction and
the establishment of the PMS-based nascent proteomics (PMSNP) method
for efficient capture and analysis of nascent proteins. The modification
efficiency of puromycin groups on silica microspheres reached 91.8%
through the click reaction. After the optimization and simplification
of PMSNP, more than 3500 and 3900 nascent proteins were rapidly identified
in HeLa cells and mouse brains within 13.5 h, respectively. The PMSNP
method was successfully applied to explore changes in the translation
process in a biological stress model, namely, the lipopolysaccharide-stimulated
HeLa cells. Biological functional analyses revealed the unique characters
of the nascent proteomes and exhibited the superiority of the PMSNP
in the identification of low abundance and secreted nascent proteins,
thus demonstrating the sensitivity and immediacy of the PMSNP method.