Proteome and Phosphoproteome Revealed Unique Features of Mouse Embryonic Stem Cells

摘要

Embryonic stem (ES) cells are pluripotent and capable of self-renewal and, therefore hold promise for cell-based regenerative medicine. For a thorough understanding of the molecular mechanisms underlying ES cell pluripotency and differentiation, studies focused on a more comprehensive omics view of human or mouse ES cells ranging from their transcriptome to proteome. The total cell lysate was trypsin digested and fractionated by SAX followed TiO2 enrichment (SAX/TiO2). Each fraction was separated on 1D-LC-MS/MS. For antibody-based purification, the cell lysate was enriched for phosphorylated serine/threonine proteins (anti-pS/T enrichment). Meanwhile, the cells were harvested and subcellular fractionation was applied to reduce the complexity. The two resulting fractions (cytosol and nuclear) were digested and separated on online yin-yang multiple dimensional liquid chromatography (MDLC), followed by high-resolution LTQ-Orbitrap mass spectrometry identification. Extensive bioinformatic analysis was applied to integrated transcriptome, proteome and modificome, aiming to reveal the unique feature of ES cells in different levels. Based on multiplexed strategies and high-coverage identification of peptide mixtures, we detected 4581 proteins and high confident 3970 distinct phosphosites from 1642 phosphoproteins. Notably, some prominent phosphorylated stem cell markers with novel phosphosites were identified by mass spectrometry for the first time. To our knowledge, this is the largest global phosphorylation characterization of mES cells. Function annotation to the phosphoproteins showed that these proteins are acting in various manners as regulation factors in DNA and RNA related biological pathways. We also investigated the expression correlation between mRNA and protein phosphorylation events in mES. Furthermore, we constructed the database containing transcriptome, proteome and modificome of stem cell to facilitate the understanding of the mechanism of its pluripotency.