Mass spectrometry-based identification of extracellular domains of cell surface N-glycoproteins: defining the accessible surfaceome for immunophenotyping stem cells and their derivatives

Chelsea M. Fujinaka, Matthew Waas, Rebekah L. Gundry

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages57-78
Number of pages22
DOIs
StatePublished - Jan 1 2018

Publication series

NameMethods in Molecular Biology
Volume1722
ISSN (Print)1064-3745

Fingerprint

Immunophenotyping
Membrane Glycoproteins
Mass Spectrometry
Stem Cells
Antibodies
Regenerative Medicine
Cell Lineage
Computational Biology
Epitopes
Glycoproteins
Membrane Proteins
Proteins

Keywords

  • Barcode
  • Cell surface
  • Cell surface protein atlas
  • Epitope selection
  • Extracellular domain
  • Immunophenotyping
  • Mass spectrometry
  • N-glycoproteins
  • Proteomics

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Mass spectrometry-based identification of extracellular domains of cell surface N-glycoproteins : defining the accessible surfaceome for immunophenotyping stem cells and their derivatives. / Fujinaka, Chelsea M.; Waas, Matthew; Gundry, Rebekah L.

Methods in Molecular Biology. Humana Press Inc., 2018. p. 57-78 (Methods in Molecular Biology; Vol. 1722).

Research output: Chapter in Book/Report/Conference proceedingChapter

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