Mass Spectrometry-Based Identification of Extracellular Domains of Cell Surface N-Glycoproteins: Defining the Accessible Surfaceome for Immunophenotyping Stem Cells and Their Derivatives

Abstract

Human stem cells and their progeny are valuable for a variety of research applications and have the potential to revolutionize approaches to regenerative medicine. However, we currently have limited tools to permit live isolation of homogeneous populations of cells apt for mechanistic studies or cellular therapies. While these challenges can be overcome through the use of immunophenotyping based on accessible cell surface markers, the success of this process depends on the availability of reliable antibodies and well-characterized markers, which are lacking for most stem cell lineages. This chapter outlines an iterative process for the development of new cell surface marker barcodes for identifying and selecting stem cell derived progeny of specific cell types, subtypes, and maturation stages, where antibody-independent identification of cell surface proteins is achieved using a modern chemoproteomic approach to specifically identify N-glycoproteins localized to the cell surface. By taking advantage of a large repository of available cell surfaceome data, proteins that are unlikely to confer cell type specificity can be rapidly eliminated from consideration. Subsequently, targeted quantitation by mass spectrometry can be used to refine candidates of interest, and a bioinformatic visualization tool is key to mapping experimental data to candidate protein sequences for the purpose of epitope selection during the antibody development phase. Overall, the process of developing cell surface barcodes for immunophenotyping is iterative and can include multiple rounds of discovery, refinement, and validation depending on the phenotypic resolution required.

Importance of evaluating protein glycosylation in pluripotent stem cell-derived cardiomyocytes for research and clinical applications

“…Finally, we have identified > 650 cell surface N- glycoproteins on hPSC-CM collected from days 20–100 of differentiation (published and unpublished) using the cell surface capture (CSC) technology [ 29 , 115 ]. The CSC approach enriches for extracellular N- glycopeptides and can be used to identify sites of glycosylation, but not the glycan moiety.…”

Assessment of Streptavidin Bead Binding Capacity to Improve Quality of Streptavidin-based Enrichment Studies

“…Cell surface capture (CSC), a chemoproteomic strategy for the identification of cell surface N -glycoproteins, was performed as we previously described in detail. − To evaluate the correlation between the variation in the binding capacity and the proteomics results, we performed CSC on RPMI 1788 cells, starting with 1000 μg of peptide pre-enrichment, and used 100 μL of total bead volume each of the four different lots of GenScript streptavidin MagBeads catalog no. L00424 (lots C44251904, C44241809, C44261906, C44242003), Cytiva Sera-Mag SpeedBeads neutravidin-coated magnetic beads (catalog no.…”

“…65001; lot 00945048) for the enrichment of N -glycopeptides, whereas all other steps were performed identically. Peptides were analyzed using an Orbitrap Fusion Lumos or Orbitrap Exploris 480 instrument (Thermo Fisher Scientific), and data were processed with ProteomeDiscoverer 2.4 (Thermo Fisher Scientific), implementing the Sequest and MSFragger search algorithms followed by Percolator for postsearch validation, as previously described. − All methodological details for the MS analysis are described in the Supporting Information.…”

The power of proteomics to monitor senescence-associated secretory phenotypes and beyond: toward clinical applications

“…CSC has been extensively applied in creating cell-type specific surfaceome databases for humans and mice, such as the Cell Surface Protein Atlas [134]. Surfaceomic technology has also been widely utilized in the areas of cancer, stem cells and immunology [131,[135][136][137]. The discovery of robust specific senescent cell-surface markers would open the door to novel drug and immunotherapeutic approaches to eliminate senescent cells and new biomarkers for senescent cell burden.…”