scholarly journals Cell-surface tethered promiscuous biotinylators enable small-scale surface proteomics of human exosomes

2021 ◽  
Author(s):  
Lisa L Kirkemo ◽  
Susanna Elledge ◽  
Jiuling Yang ◽  
James Robert Byrnes ◽  
Jeff Edward Glasgow ◽  
...  
Keyword(s):  
Cell Surface ◽  
High Surface ◽  
Surface Proteins ◽  
Small Scale ◽  
Myc Oncogene ◽  
Cell Surface Biotinylation ◽  
Hrp Method ◽  
Surface Proteome ◽  
Scale Surface

Characterization of cell surface proteome differences between cancer and healthy cells is a valuable approach for the identification of novel diagnostic and therapeutic targets. However, selective sampling of surface proteins for proteomics requires large samples (>10e7 cells) and long labeling times. These limitations preclude analysis of material-limited biological samples or the capture of rapid surface proteomic changes. Here, we present two labeling approaches to tether exogenous peroxidases (APEX2 and HRP) directly to cells, enabling rapid, small-scale cell surface biotinylation without the need to engineer cells. We used a novel lipidated DNA-tethered APEX2 (DNA-APEX2), which upon addition to cells promoted cell agnostic membrane-proximal labeling. Alternatively, we employed horseradish peroxidase (HRP) fused to the glycan binding domain of wheat germ agglutinin (WGA-HRP). This approach yielded a rapid and commercially inexpensive means to directly label cells containing common N-Acetylglucosamine (GlcNAc) and sialic acid glycans on their surface. The facile WGA-HRP method permitted high surface coverage of cellular samples and enabled the first comparative surface proteome characterization of cells and cell-derived exosomes, leading to the robust quantification of 1,020 cell and exosome surface proteins. We identified a newly-recognized subset of exosome-enriched markers, as well as proteins that are uniquely upregulated on Myc oncogene-transformed prostate cancer exosomes. These two cell-tethered enzyme surface biotinylation approaches are highly advantageous for rapidly and directly labeling surface proteins across a range of material-limited sample types.

scholarly journals Comprehensive cell surface proteomics defines markers of classical, intermediate and non-classical monocytes

2020 ◽  
Author(s):  
Benjamin J. Ravenhill ◽  
Lior Soday ◽  
Jack Houghton ◽  
Robin Antrobus ◽  
Michael P. Weekes
Keyword(s):  
Cell Surface ◽  
Surface Proteins ◽  
Monocyte Subset ◽  
Protein Markers ◽  
Phenotypic Differences ◽  
Tandem Mass Tag ◽  
Cell Surface Biotinylation ◽  
Cd16 Expression ◽  
Surface Proteome ◽  
Classical Monocytes

AbstractMonocytes are a critical component of the cellular innate immune system, and can be subdivided into classical, intermediate and non-classical subsets on the basis of surface CD14 and CD16 expression. Classical monocytes play the canonical role of phagocytosis, and account for the majority of circulating cells. Intermediate and non-classical cells are known to exhibit varying levels of phagocytosis and cytokine secretion, and are differentially expanded in certain pathological states. Characterisation of cell surface proteins expressed by each subset is informative not only to improve understanding of phenotype, but may also provide biological insights into function. Here we use highly multiplexed Tandem-Mass-Tag (TMT)-based mass spectrometry with selective cell surface biotinylation to characterise the classical monocyte surface proteome, then interrogate the phenotypic differences between each monocyte subset to identify novel protein markers.

The E-selectin-ligand ESL-1 is located in the Golgi as well as on microvilli on the cell surface

1997 ◽  
Vol 110 (6) ◽  
pp. 687-694 ◽  
Author(s):  
M. Steegmaier ◽  
E. Borges ◽  
J. Berger ◽  
H. Schwarz ◽  
D. Vestweber
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Surface Proteins ◽  
Intact Cells ◽  
Fgf Receptor ◽  
Cell Surface Biotinylation ◽  
Cell Surface Staining ◽  
Surface Staining ◽  
Selectin Ligand ◽  
Golgi Protein

Neutrophils and subsets of lymphocytes bind to E-selectin, a cytokine inducible adhesion molecule on endothelial cells. The E-selectin-ligand-1 (ESL-1) is a high affinity glycoprotein ligand which participates in the binding of mouse myeloid cells to E-selectin. The sequence of mouse ESL-1 is highly homologous to the cysteine rich FGF receptor (CFR) in chicken and the rat Golgi protein MG160. We have analysed the subcellular distribution of ESL-1 by indirect immunofluorescence, flow cytometry, various biochemical techniques and by immunogold scanning electron microscopy. We could localize ESL-1 in the Golgi as well as on the cell surface of 32Dc13 cells and neutrophils. Cell surface staining was confirmed by cell surface biotinylation and by cell surface immunoprecipitations in which antibodies only had access to surface proteins on intact cells. In addition, ESL-1(high) and ESL-1(low) expressing cells, sorted by flow cytometry, gave rise to high and low immunoprecipitation signals for ESL-1, respectively. Based on immunogold labeling of intact cells, we localized ESL-1 on microvilli of 32Dc13 cells and of the lymphoma cell line K46. Quantitative evaluation determined 80% of the total labeling for ESL-1 on microvilli of K46 cells while 69% of the labeling for the control antigen B220 was found on the planar cell surface. These data indicate that ESL-1 occurs at sites on the leukocyte cell surface which are destined for the initiation of cell contacts to the endothelium.

scholarly journals Quantitative comparative analysis of human erythrocyte surface proteins between individuals from two genetically distinct populations

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Benjamin J. Ravenhill ◽  
Usheer Kanjee ◽  
Ambroise Ahouidi ◽  
Luis Nobre ◽  
James Williamson ◽  
...  
Keyword(s):  
Cell Surface ◽  
Critical Role ◽  
Surface Proteins ◽  
Human Populations ◽  
Cell Surface Proteins ◽  
Systematic Analysis ◽  
Tandem Mass Tag ◽  
The Uk ◽  
Analysis Of Variation ◽  
Surface Proteome

Abstract Red blood cells (RBCs) play a critical role in oxygen transport, and are the focus of important diseases including malaria and the haemoglobinopathies. Proteins at the RBC surface can determine susceptibility to disease, however previous studies classifying the RBC proteome have not used specific strategies directed at enriching cell surface proteins. Furthermore, there has been no systematic analysis of variation in abundance of RBC surface proteins between genetically disparate human populations. These questions are important to inform not only basic RBC biology but additionally to identify novel candidate receptors for malarial parasites. Here, we use ‘plasma membrane profiling’ and tandem mass tag-based mass spectrometry to enrich and quantify primary RBC cell surface proteins from two sets of nine donors from the UK or Senegal. We define a RBC surface proteome and identify potential Plasmodium receptors based on either diminished protein abundance, or increased variation in RBCs from West African individuals.

scholarly journals Characterization of small-scale surface topography using transmission electron microscopy

2018 ◽  
Vol 6 (4) ◽  
pp. 045004 ◽  
Author(s):  
Subarna R Khanal ◽  
Abhijeet Gujrati ◽  
Sai Bharadwaj Vishnubhotla ◽  
Pawel Nowakowski ◽  
Cecile S Bonifacio ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Surface Topography ◽  
Small Scale ◽  
Transmission Electron ◽  
Scale Surface

scholarly journals Characterization of T-cell surface proteins bound by heterologous antisera to antigen-specific T-cell products.

1981 ◽  
Vol 78 (10) ◽  
pp. 6411-6415 ◽  
Author(s):  
R. E. Cone ◽  
R. W. Rosenstein ◽  
J. H. Murray ◽  
G. M. Iverson ◽  
W. Ptak ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
Surface Proteins ◽  
Cell Surface Proteins

scholarly journals Multi-omics analysis of AML cells treated with azacitidine reveals highly variable cell surface proteome remodeling

2018 ◽  
Author(s):  
Kevin K Leung ◽  
Aaron Nguyen ◽  
Tao Shi ◽  
Lin Tang ◽  
Xiaochun Ni ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Protein Level ◽  
Dna Methyltransferase ◽  
Immune Defense ◽  
Surface Proteins ◽  
Gene Set Enrichment Analysis ◽  
Surface Proteome ◽  
The Impact ◽  
Cell Surface Proteome

AbstractMyelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are diseases of abnormal hematopoietic differentiation with aberrant epigenetic alterations. Azacitidine (AZA) is a DNA methyltransferase inhibitor (DNMTi) widely used to treat MDS and AML, yet the impact of AZA on the cell surface proteome has not been defined. To identify potential therapeutic targets for use in combination with AZA in AML patients, we investigated the effects of AZA treatment on four AML cell lines (KG1a, HL60, HNT34, and AML193), representing different stages of differentiation. The effect of AZA treatment on these cell lines was characterized at three levels: the DNA methylome (methylation array), the transcriptome (gene expression array), and the cell surface proteome (glycoprotein capture with SILAC labeling). Untreated AML cell lines showed substantial overlap in their methylomes, transcriptomes, and cell surface proteomes. AZA treatment globally reduced DNA methylation in all cell lines, but changes in the transcriptome and surface proteome were subtle and differed among the cell lines. Transcriptome analysis identified five commonly up-regulated coding genes upon AZA treatment in all four cell lines, TRPM4 being the only gene encoding a surface protein, and surface proteomics analysis found no commonly regulated proteins. Gene Set Enrichment Analysis (GSEA) of differentially-regulated RNA and surface proteins showed a decrease in metabolism pathways and an increase in immune defense response pathways. As such, AZA treatment in four AML cell lines had diverse effects at the individual gene and protein level, but converged to regulation of metabolism and immune response at the pathway level. Given the heterogeneous response of AZA in the four cell lines at the gene and protein level, we discuss potential therapeutic strategies for combinations with AZA.

scholarly journals Coupling of Cell Surface Biotinylation and SILAC-Based Quantitative Proteomics Identified Myoferlin as a Potential Therapeutic Target for Nasopharyngeal Carcinoma Metastasis

2021 ◽  
Vol 9 ◽  
Author(s):  
Maoyu Li ◽  
Fang Peng ◽  
Guoqiang Wang ◽  
Xujun Liang ◽  
Meiying Shao ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Cell Surface ◽  
Therapeutic Target ◽  
Surface Proteins ◽  
Differentially Expressed ◽  
Attractive Potential ◽  
Potential Therapeutic Target ◽  
Cell Surface Proteins ◽  
Mesenchymal Transition ◽  
Cell Surface Biotinylation

Distant metastasis is a major cause of treatment failure in nasopharyngeal carcinoma (NPC) patients. Cell surface proteins represent attractive targets for cancer diagnosis or therapy. However, the cell surface proteins associated with NPC metastasis are poorly understood. To identify potential therapeutic targets for NPC metastasis, we isolated cell surface proteins from two isogenic NPC cell lines, 6-10B (low metastatic) and 5-8F (highly metastatic), through cell surface biotinylation. Stable isotope labeling by amino acids in cell culture (SILAC) based proteomics was applied to comprehensively characterize the cell surface proteins related with the metastatic phenotype. We identified 294 differentially expressed cell surface proteins, including the most upregulated protein myoferlin (MYOF), two receptor tyrosine kinases(RTKs) epidermal growth factor receptor (EGFR) and ephrin type-A receptor 2 (EPHA2) and several integrin family molecules. These differentially expressed proteins are enriched in multiple biological pathways such as the FAK-PI3K-mTOR pathway, focal adhesions, and integrin-mediated cell adhesion. The knockdown of MYOF effectively suppresses the proliferation, migration and invasion of NPC cells. Immunohistochemistry analysis also showed that MYOF is associated with NPC metastasis. We experimentally confirmed, for the first time, that MYOF can interact with EGFR and EPHA2. Moreover, MYOF knockdown could influence not only EGFR activity and its downstream epithelial–mesenchymal transition (EMT), but also EPHA2 ligand-independent activity. These findings suggest that MYOF might be an attractive potential therapeutic target that has double effects of simultaneously influencing EGFR and EPHA2 signaling pathway. In conclusion, this is the first study to profile the cell surface proteins associated with NPC metastasis and provide valuable resource for future researches.

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