A cell surfaceome map for immunophenotyping and sorting pluripotent stem cells.

Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N-glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations.

The mouse C2C12 myoblast cell surface N-linked glycoproteome: identification, glycosite occupancy, and membrane orientation.

Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and beta-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes.

Expanding the mouse embryonic stem cell proteome: combining three proteomic approaches.

The current study used three different proteomic strategies, which differed by their extent of intact protein separation, to examine the proteome of a pluripotent mouse embryonic stem cell line, R1. Proteins from whole-cell lysates were subjected either to 2-D-LC, or 1-DE, or were unfractionated prior to enzymatic digestion and subsequent analysis by MS. The results yielded 1895 identified non-redundant proteins and, for 128 of these, the specific isoform could be determined based on detection of an isoform-specific peptide. When compared with two previously published proteomic studies that used the same cell line, the current study reveals 612 new proteins.