N‐glycoprotein surfaceomes of four developmentally distinct mouse cell types

Abstract

Purpose Detailed knowledge of cell surface proteins present during early embryonic development remains limited for most cell lineages. Due to the relevance of cell surface proteins in their functional roles controlling cell signaling and their utility as accessible, non-genetic markers for cell identification and sorting, the goal of this study was to provide new information regarding the cell surface proteins present during early mouse embryonic development. Experimental Design Using the Cell Surface Capture Technology, the cell surface N-glycoproteomes of three cell lines and one in vitro differentiated cell type representing distinct cell fates and stages in mouse embryogenesis were assessed. Results Altogether, more than 600 cell surface N-glycoproteins were identified represented by >5500 N-glycopeptides. Conclusions and Clinical Relevance The development of new, informative cell surface markers for the reliable identification and isolation of functionally defined subsets of cells from early developmental stages will advance the use of stem cell technologies for mechanistic developmental studies, including disease modeling and drug discovery.

Concise Review: Cell Surface N-Linked Glycoproteins as Potential Stem Cell Markers and Drug Targets

“…This poses challenges to obtaining robust MS‐based quantitation, which ideally relies on multiple peptides within a protein. Nevertheless, we have successfully performed quantitative comparisons between mESCs and mouse extraembryonic endoderm cells using stable isotope labeling with amino acids in cell culture (SILAC) . In these experiments, SILAC ratios proved consistent with flow‐cytometry analyses and, when available, were consistent with published reports.…”

“…We confirmed that CD172a could be detected on mPSC-CM by using an alternative antibody to the one used in the original study, highlighting a common challenge for antibody-based studies, wherein the absence of a positive signal does not confirm absence of the protein, but could indicate that a conformational or posttranslational modification masks the epitope required for antibody recognition. Moreover, in our CSC technology analyses of mPSC, hPSC, and their cardiogenic derivatives ( [20,21,30] and unpublished data), we have identified previously reported cell surface markers for stem cell-derived cardiac progenitors and cardiomyocytes-including ROR2, KDR, VCAM1 (CD106), ALCAM (CD166), ANPEP (CD13), PDGFRa (CD140a), EMI-LIN2, KIT (CD117), and CD172a-within a handful of experiments without the use of antibodies. Comparisons of these data within the published CSPA that have been augmented with unpublished analyses of primary hepatocytes, human iPSC (hiPSC)-derived hepatic progenitors, pigmented retinal epithelial cells, blood cells, and cardiac fibroblasts quickly reveal that ROR2, VCAM1, ALCAM, ANPEP, PDGFRa, and SIRPA are observed across many different noncardiomyogenic cell types, which may impact their utility for identification of a single or defined cell population.…”

“…In this strategy, the experimental output confirms the occupancy of individual N ‐glycosylation sites of identified proteins and thereby confirms extracellular domains. Complementary variations of this approach that rely on cysteine‐ or lysine‐containing peptides have also been described , and quantitative assessments using label‐free and label‐based methods are beginning to be used , . Although the CSC technology method importantly offers the ability to view a highly specific “snapshot” of the cell surface at a particular time or stage—and thereby is advantageous over predictive approaches or those that rely on more generic membrane protein‐enrichment strategies—the extensive sample‐handling steps involved in the workflow ultimately result in the requirement for large amounts of starting material (e.g., 30 million to 100 million cells per experiment).…”

Quantitative Assessment of Sialo‐Glycoproteins and N‐Glycans during Cardiomyogenic Differentiation of Human Induced Pluripotent Stem Cells

“…The vast majority of cell‐surface proteins are N‐glycosylated, and often the extreme ends of these glycan chains contain N ‐acetylneuraminic acids (sialic acids), thus making sialic acids ideal targets for capturing cell‐surface proteins . Various reports have described the selective capture and enrichment of surface‐exposed sialo‐glycoproteins based on periodate oxidation of the glycan into an aldehyde, which can subsequently react with hydrazides or aminooxy compounds carrying affinity tags . By using this approach in combination with label‐free quantification, Boheler et al.…”

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

“…In Subheading 3.8, we show how to prioritize epitopes for marker and antibody development. We have used this strategy to identify markers and to generate new antibodies for a variety of stem cells and their progeny, including: selecting pluripotent stem cell-derived hepatocytes [28] and cardiomyocytes ([29] and in preparation ); isolating induced pluripotent stem cells reprogrammed from mouse fibroblasts [30]; and identifying proteins in human pluripotent stem cells that can be targeted by small molecules for the purpose of eliminating tumorigenic cells [25]. Although our previous work and this chapter have focused on stem cells, the approach is equally applicable to other mammalian cell types, as demonstrated by others [16, 20, 24, 31–33], and variations of this approach that rely on cysteine or lysine containing peptides have also been described [31].…”