Proteomic Response Fingerprinting (ProReF) for Rapid Identification of Protein Targets for Chinese Medicine

Abstract

Chinese medicine (CM) has been used as the clinical therapies for thousands of years in China. The chemical complexity of Chinese medicines makes it difficult to dissect the molecular mechanisms by single-target based approaches. Proteomics is a recently emerged powerful technology for simultaneous detection of multiple protein targets in the cells and tissues. Proteomic response fingerprinting (ProReF) approach uses 2-D SDS-PAGE and MALDI-TOF mass spectrometry to examine the effects of Chinese medicines on the expression levels of various cellular proteins in the cell cultures or animal organs/tissues. In practical, following the treatments with/without Chinese medicines, the protein lysates are analyzed by two-dimensional gel electrophoresis (2-DE). Based on the change of signal intensity, specific protein spots are selected and subsequently identified by mass spectrometry (MS) techniques. The selected proteins are systematically identified and quantitated as the cellular responses to CM. Proteomic information is important for the investigation of target proteins and relevant signaling pathways, thereby facilitating the understanding on the mechanism of action of CM. Here we reviewed the application of proteomics to discover the molecular mechanisms underlying the pharmacological activities of CM formula. We discussed the basic principles and useful protocols for proteomic analyses. We provided detailed procedures for sample preparation (control and CM-treated group), 2-D gel electrophoresis, image comparison, spot excision, trypsin digestion, mass spectrometry identification, and bioinformatics analysis. We also discussed the cellular and molecular approaches for characterizing the regulation of protein expression by active compounds. We anticipate that the readers will easily adapt these procedures for their discoveries of the target proteins for the active CM compounds of interest.