Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Proteome Quantitation of Mouse Embryonic Stem Cells to a Depth of

Embryonic stem (ES) cells are pluripotent cells isolated from mammalian preimplantation embryos. They are ca-pable of differentiating into all cell types and therefore hold great promise in regenerative medicine. Here we show that murine ES cells can be fully SILAC (stable isotope labeling by amino acids in cell culture)-labeled when grown feeder-free during the last phase of cell cul-ture. We fractionated the SILAC-labeled ES cell proteome by one-dimensional gel electrophoresis and by isoelectric focusing of peptides. High resolution analysis on a linear ion trap-orbitrap instrument (LTQ-Orbitrap) at sub-ppm mass accuracy resulted in confident identification and quantitation of more than 5,000 distinct proteins. This is the largest quantified proteome reported to date and con-tains prominent stem cell markers such as OCT4, NANOG