Characterizing the Human Embryonic Stem Cell Proteome – Life with an ETD-Enabled Orbitrap
Douglas Phanstiel, David Good, Joshua Coon1
1Chemistry, University of Wisconsin, Madison
We describe the use of new mass spectrometry technology – an ETD-enabled Orbitrap – to map and quantify the human ES cell proteome. The new instrument allows for the implementation of multiple dissociation methods, i.e., ion trap CAD, beam-type CAD (HCD), and ETD, and for the automated selection of each in a real-time based on multiple precursor attributes (i.e., data-dependent decision tree). Protein quantification is readily accomplished through use of isotopic labels – either SILAC or iTRAQ. The instrument will likewise propel top-down proteomics as acquisition of ETD-MS/MS spectra in the high resolving power Orbitrap allows for direct analysis of intact proteins on a sub-second timescale with ~ 300 ppb mass accuracies. Such mass accuracies are used to directly annotate ETD tandem mass spectral peaks with ion type and chemical composition. We demonstrate these and many other aspects of the instrument on a variety of applications involving human ES cells, differentiating human ES cells, and induced-pluripotent cells.
0 Items 