Effective Method for Accurate and Sensitive Quantitation of Rapid Changes of Newly Synthesized Proteins

Protein
synthesis is quickly and tightly regulated in cells to
adapt to the ever-changing extracellular and intracellular environment.
Accurate quantitation of rapid protein synthesis changes can provide
insights into protein functions and cellular activities, but it is
very challenging to achieve because of the lack of effective analysis
methods. Here, we developed an effective mass spectrometry-based method
named quantitative O-propargyl-puromycin tagging (QOT) by integrating
O-propargyl-puromycin (OPP) labeling, bioorthogonal chemistry, and
multiplexed proteomics for global and quantitative analysis of rapid
protein synthesis. The current method enables us to accurately quantitate
rapid changes of newly synthesized proteins because, unlike amino
acids and their analogs, OPP can be utilized by the ribosome immediately
without being activated and conjugated to tRNA, and thus cell starvation
or pretreatment is not required. This method was applied to quantitate
rapid changes of protein synthesis in THP-1 macrophages treated with
lipopolysaccharide (LPS). For 15-min labeling, >3000 proteins were
quantitated, and the synthesis of 238 proteins was significantly altered,
including transcription factors and cytokines. The results demonstrated
that protein synthesis was modulated to facilitate protein secretion
in macrophages in response to LPS. Considering the importance of protein
synthesis, this method can be extensively applied to investigate rapid
changes of protein synthesis in the biological and biomedical research
fields.