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.