{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/359987"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/359987","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"An unbiased proteomic approach to identifying cell surface markers in neutrophils","abstract":"Introduction: Efferocytosis refers to the engulfment of dead cells by phagocytes such as neutrophils and macrophages and culminates in the beneficial removal of inflammatory cargo and auto-reactive material. While many of the mechanisms involved in efferocytosis have been described, the critically important ‘apoptotic-cell-associated molecular patterns’ have yet to be identified. Methods: Human whole blood samples were obtained from healthy volunteers. Neutrophils were isolated and subjected to a temperature shift to facilitate a wave of synchronised apoptosis. The plasma membrane proteome was assessed using tandem-mass-tag liquid chromatography-mass spectrometry (TMT-LC/MS) of apoptotic and time- and donor- matched non-apoptotic neutrophils, which allowed the identification of the plasma membrane proteins expressed or downregulated during apoptosis. In parallel, a method was developed to quantify efferocytosis. Briefly, apoptotic neutrophils or genome-modified neutrophil-like cells overexpressing the proteins identified to be up- regulated on apoptotic neutrophils, were fed to the monocytic cell line THP-1, which had been previously primed with phorbol-12-myristate-13-acetate (PMA) to acquire the functional properties of human macrophages. Efferocytosis was assessed by confocal microscopy and flow cytometry. Results: The late-stage apoptotic neutrophil plasma membrane profile identified several protein targets to be either up- or downregulated. The expression kinetics over time of ‘targets’ were then validated using flow cytometry. TIMD-4, a never previously described protein in (human) neutrophils, is actively up- regulated in preparation for apoptosis, and links to the phagocyte’s recognition and removal abilities of apoptotic cells, suggesting that TIMD-4 is a novel ‘eat me’ signal produced by apoptotic neutrophils. Conclusions: TMT-LC/MS can be successfully used to determine functionally relevant changes in the neutrophil plasma membrane, and has provided novel insights into the processes underlying efferocytosis of human neutrophils.","abstract_html":"Introduction: Efferocytosis refers to the engulfment of dead cells by phagocytes such as neutrophils and macrophages and culminates in the beneficial removal of inflammatory cargo and auto-reactive material. While many of the mechanisms involved in efferocytosis have been described, the critically important ‘apoptotic-cell-associated molecular patterns’ have yet to be identified. Methods: Human whole blood samples were obtained from healthy volunteers. Neutrophils were isolated and subjected to a temperature shift to facilitate a wave of synchronised apoptosis. The plasma membrane proteome was assessed using tandem-mass-tag liquid chromatography-mass spectrometry (TMT-LC/MS) of apoptotic and time- and donor- matched non-apoptotic neutrophils, which allowed the identification of the plasma membrane proteins expressed or downregulated during apoptosis. In parallel, a method was developed to quantify efferocytosis. Briefly, apoptotic neutrophils or genome-modified neutrophil-like cells overexpressing the proteins identified to be up- regulated on apoptotic neutrophils, were fed to the monocytic cell line THP-1, which had been previously primed with phorbol-12-myristate-13-acetate (PMA) to acquire the functional properties of human macrophages. Efferocytosis was assessed by confocal microscopy and flow cytometry. Results: The late-stage apoptotic neutrophil plasma membrane profile identified several protein targets to be either up- or downregulated. The expression kinetics over time of ‘targets’ were then validated using flow cytometry. TIMD-4, a never previously described protein in (human) neutrophils, is actively up- regulated in preparation for apoptosis, and links to the phagocyte’s recognition and removal abilities of apoptotic cells, suggesting that TIMD-4 is a novel ‘eat me’ signal produced by apoptotic neutrophils. Conclusions: TMT-LC/MS can be successfully used to determine functionally relevant changes in the neutrophil plasma membrane, and has provided novel insights into the processes underlying efferocytosis of human neutrophils.","abstract_has_math":false,"creators":["Subburayalu, Julien"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Summers, Charlotte"],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-07-03","date_published":"2022-07-03","updated_at":"2026-07-22T22:24:31Z","subjects":["Apoptosis","Eat-me signal","Efferocytosis","HL-60","Macrophage","Neutrophil","Plasma membrane profiling","Resolution of Inflammation","THP-1","TIMD-4"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3f6a1904-155c-47e5-8ec1-3100edc44155/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000192430558"],"render_values":[{"text":"0000-0001-9243-0558","href":"https://orcid.org/0000-0001-9243-0558","code":true}]}]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.103277","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Summers, Charlotte"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["AstraZeneca/MedImmune (RCAG/891) NIHR Cambridge Biomedical Research Centre German Academic Scholarship Foundation (Studienstiftung des deutschen Volkes)"]},{"key":"dc:creator","label":"Author","values":["Subburayalu, Julien"]},{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000192430558"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2022-07-03"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/359987"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Apoptosis","Eat-me signal","Efferocytosis","HL-60","Macrophage","Neutrophil","Plasma membrane profiling","Resolution of Inflammation","THP-1","TIMD-4"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3f6a1904-155c-47e5-8ec1-3100edc44155/download","https://www.rioxx.net/licenses/all-rights-reserved/"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2026-11-09"]},{"key":"dc:rights.embargotype","label":"Dc Rights Embargotype","values":["embargo"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.103277"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/53a38d4f-0f77-4f15-9697-9c93b44ab76f/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Introduction: Efferocytosis refers to the engulfment of dead cells by phagocytes such as neutrophils and macrophages and culminates in the beneficial removal of inflammatory cargo and auto-reactive material. While many of the mechanisms involved in efferocytosis have been described, the critically important ‘apoptotic-cell-associated molecular patterns’ have yet to be identified. Methods: Human whole blood samples were obtained from healthy volunteers. Neutrophils were isolated and subjected to a temperature shift to facilitate a wave of synchronised apoptosis. The plasma membrane proteome was assessed using tandem-mass-tag liquid chromatography-mass spectrometry (TMT-LC/MS) of apoptotic and time- and donor- matched non-apoptotic neutrophils, which allowed the identification of the plasma membrane proteins expressed or downregulated during apoptosis. In parallel, a method was developed to quantify efferocytosis. Briefly, apoptotic neutrophils or genome-modified neutrophil-like cells overexpressing the proteins identified to be up- regulated on apoptotic neutrophils, were fed to the monocytic cell line THP-1, which had been previously primed with phorbol-12-myristate-13-acetate (PMA) to acquire the functional properties of human macrophages. Efferocytosis was assessed by confocal microscopy and flow cytometry. Results: The late-stage apoptotic neutrophil plasma membrane profile identified several protein targets to be either up- or downregulated. The expression kinetics over time of ‘targets’ were then validated using flow cytometry. TIMD-4, a never previously described protein in (human) neutrophils, is actively up- regulated in preparation for apoptosis, and links to the phagocyte’s recognition and removal abilities of apoptotic cells, suggesting that TIMD-4 is a novel ‘eat me’ signal produced by apoptotic neutrophils. Conclusions: TMT-LC/MS can be successfully used to determine functionally relevant changes in the neutrophil plasma membrane, and has provided novel insights into the processes underlying efferocytosis of human neutrophils."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["07ed17e8fa584e0f0c19ec1f0d86709b","87eda9de84448d1f82354d60eee3eb5f"]},{"key":"dc:title","label":"Title","values":["An unbiased proteomic approach to identifying cell surface markers in neutrophils"]}]}],"canonical_facts":{"dc:contributor.advisor":["Summers, Charlotte"],"dc:contributor.sponsor":["AstraZeneca/MedImmune (RCAG/891) NIHR Cambridge Biomedical Research Centre German Academic Scholarship Foundation (Studienstiftung des deutschen Volkes)"],"dc:creator":["Subburayalu, Julien"],"dc:creator.authoridentifier":["0000000192430558"],"dc:date.issued":["2022-07-03"],"dc:description.abstract":["Introduction: Efferocytosis refers to the engulfment of dead cells by phagocytes such as neutrophils and macrophages and culminates in the beneficial removal of inflammatory cargo and auto-reactive material. While many of the mechanisms involved in efferocytosis have been described, the critically important ‘apoptotic-cell-associated molecular patterns’ have yet to be identified. Methods: Human whole blood samples were obtained from healthy volunteers. Neutrophils were isolated and subjected to a temperature shift to facilitate a wave of synchronised apoptosis. The plasma membrane proteome was assessed using tandem-mass-tag liquid chromatography-mass spectrometry (TMT-LC/MS) of apoptotic and time- and donor- matched non-apoptotic neutrophils, which allowed the identification of the plasma membrane proteins expressed or downregulated during apoptosis. In parallel, a method was developed to quantify efferocytosis. Briefly, apoptotic neutrophils or genome-modified neutrophil-like cells overexpressing the proteins identified to be up- regulated on apoptotic neutrophils, were fed to the monocytic cell line THP-1, which had been previously primed with phorbol-12-myristate-13-acetate (PMA) to acquire the functional properties of human macrophages. Efferocytosis was assessed by confocal microscopy and flow cytometry. Results: The late-stage apoptotic neutrophil plasma membrane profile identified several protein targets to be either up- or downregulated. The expression kinetics over time of ‘targets’ were then validated using flow cytometry. TIMD-4, a never previously described protein in (human) neutrophils, is actively up- regulated in preparation for apoptosis, and links to the phagocyte’s recognition and removal abilities of apoptotic cells, suggesting that TIMD-4 is a novel ‘eat me’ signal produced by apoptotic neutrophils. Conclusions: TMT-LC/MS can be successfully used to determine functionally relevant changes in the neutrophil plasma membrane, and has provided novel insights into the processes underlying efferocytosis of human neutrophils."],"dc:format.checksum.md5":["07ed17e8fa584e0f0c19ec1f0d86709b","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.103277"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/53a38d4f-0f77-4f15-9697-9c93b44ab76f/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/359987"],"dc:rights":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/3f6a1904-155c-47e5-8ec1-3100edc44155/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:rights.embargodate":["2026-11-09"],"dc:rights.embargotype":["embargo"],"dc:subject":["Apoptosis","Eat-me signal","Efferocytosis","HL-60","Macrophage","Neutrophil","Plasma membrane profiling","Resolution of Inflammation","THP-1","TIMD-4"],"dc:title":["An unbiased proteomic approach to identifying cell surface markers in neutrophils"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:31Z"}