Identification of mouse embryonic stem cell-associated proteins.

Over the past few years, there has been a growing interest in discovering the molecular mechanisms controlling embryonic stem cells' (ESCs) proliferation and differentiation. Proteome analysis has proven to be an effective approach to comprehensively unravel the regulatory network of differentiation. We applied a two-dimensional electrophoresis based proteomic approach followed by mass spectrometry to analyze the proteome of two mouse ESC lines, Royan B1 and D3, at 0, 6, and 16 days after differentiation initiation. Out of 97 ESC-associated proteins commonly expressed in two ESC lines, 72 proteins were identified using MALDI TOF-TOF mass spectrometry analysis. The expression pattern of four down-regulated proteins including Hspd1, Hspa8, beta-Actin, and Tpt1 were further confirmed by Western blot and immunofluorescence analyses in Royan B1 and D3 as well as two other mouse ESC lines, Royan C1 and Royan C4. Differential mRNA expression analysis of 20 genes using quantitative real-time reverse transcription PCR revealed a low correlation between mRNA and protein levels during differentiation. We also observed that the mRNA level of Tpt1 increased significantly in differentiating cells, whereas its protein level decreased. Several novel ESC-associated proteins have been presented in this study which warrants further investigation with respect to the etiology of stemness.