The subcellular proteome of mouse pluripotent embryonic stem cells
Our understanding of the biology of embryonic stem (ES) cells is deeply rooted in characterization of their transcriptomes, epigenetics and underlying gene regulatory networks. There is evidence that post-transcriptional processes such as signaling, adhesion, protein turnover and post translational modifications make a significant contribution to regulating the balance between self-renewal and differentiation, and it is therefore necessary to also characterize ES cells at the protein level. In this experiment, we used a workflow termed hyperLOPIT (hyperplexed localization of organelle proteins by isotope tagging) to characterize the subcellular distribution of proteins in a population of self-renewing E14TG2a mouse ES cells. Over 5,000 protein groups were quantified in both of the two replicates, enabling characterization of protein localization to organelles (including sub-nuclear resolution), cell surface, cytoskeleton and cytosol. The steady-state localization of transitory proteins, protein complex constituents, and signaling cascades could also be mapped.
Sample Processing Protocol
E14TG2a mouse pluripotent embryonic stem cells were cultured in conditions favoring self-renewal (medium supplemented with fetal bovine serum and LIF). Cells were harvested and lysed by ball bearing homogenization. 9 differentially enriched subcellular fractions of cytosol, membranes, cytoskeleton and macromolecular complexes were generated by iodixanol density gradient ultracentrifugation. In parallel, a proportion of cells were used to generate a chromatin enriched fraction. Proteins from the 10 fractions were extracted, digested with trypsin, and labeled with TMT 10-plex reagents. The peptide mixture was pre-fractionated into 24 fractions by high pH reversed phase LC. Each fraction was analyzed in a 2 hour run on the Orbitrap Fusion platform (Thermo Fisher Scientific) in SPS-MS3 mode, with 10 notches per MS3 event.
Data Processing Protocol
Raw data files were processed using Proteome Discoverer 1.4 (Thermo Fisher Scientific). Peptide-spectrum matching was performed against the SwissProt mouse sequence database (March 2013; 24,481 sequences) with the Mascot search algorithm (v2.3.02, Matrix Science). PSMs from Mascot were re-scored by Proteome Discoverer's 'Percolator' node. TMT 10-plex quantification was performed by Proteome Discoverer's 'Reporter Ion Quantifier' node.
Christoforou A, Mulvey CM, Breckels LM, Geladaki A, Hurrell T, Hayward PC, Naake T, Gatto L, Viner R, Arias AM, Lilley KS. A draft map of the mouse pluripotent stem cell spatial proteome. Nat Commun. 2016 Jan 12;7:9992 PubMed: 26754106