{"id":{"repo_id":"uic","oai_identifier":"oai:figshare.com:article/32995250"},"canonical_url":"https://search.dev.ndltd.org/etd/uic/oai:figshare.com:article/32995250","repository":{"repo_id":"uic","name":"University of Illinois - Chicago","base_url":"https://api.figshare.com/v2/oai"},"display":{"title":"Enhanced Proteomic Methods to Decipher How the Novel Protein SNED1 Modulates the Extracellular Matrix","abstract":"The extracellular matrix (ECM) is a complex meshwork of proteins that provides not only structural support but also biochemical cues to cells. The assembly of the ECM is a dynamic process requiring precise coordination: 1) ECM proteins are produced, post-translationally modified, and then secreted into the extracellular space, 2) once secreted, ECM proteins are organized into protein networks with the aid of the other ECM proteins and ECM receptors on the cell surface, 3) this network of proteins is further maintained and remodeled over time to carry out structural and biochemical roles affecting cell behavior. Thus, a finely tuned balance of the proteins that form and maintain the ECM is essential to health, while an imbalance can cause or accompany physiological and pathological changes, ranging from the wrinkles of the skin to fibrotic diseases and cancer metastases. Despite the significant roles of the ECM in health and disease, studying the ECM had been difficult for a long time due to its biochemically complex nature. However, my mentor, Dr. Alexandra Naba developed a proteomic pipeline tailored to study the protein composition of the ECM, which has since become the method of choice in studying the ECM. Using this method, Dr. Naba identified a novel ECM protein, SNED1, as being only present in the ECM of highly metastatic mammary tumors. Upon this discovery, Dr. Naba knocked down SNED1 in a mammary tumor cell line. When the resulting cells were intravenously injected to mimic the already extravasated tumor cells, the SNED1 knockdown cells did not result in a reduced metastasis. However, when orthotopically injected into mice, SNED1 knockdown cells resulted in fewer metastatic foci compared to the control counterparts, suggesting SNED1 is a protein promoting the invasiveness of the tumor cells. When I started my PhD, not much was known about SNED1. Previous work of the lab on the SNED1 knockout mouse model revealed that SNED1 is essential gene for development. Further work on this aspect by a lab alumna led to identification of SNED1’s involvement in neural crest cell migration. On the other hand, second harmonic generation microscopy revealed that presence of SNED1 altered the ECM organization, but the SNED1-dependent changes in the matrisome was not yet identified. Thus, I hypothesized that SNED1 modulates the ECM architecture by influencing the ECM composition. In this thesis, I focused on answering this biological question and on enhancing proteomic methods to do so, using mouse embryonic fibroblasts derived from a SNED1-knockout mice engineered to overexpress SNED1 or not. To characterize the SNED1-dependent changes in the ECM composition, I used ECM proteomics to compare the compositions of ECMs made by cells overexpressing or not SNED1. To additionally increase the sequence coverage of the ECM proteins, I developed an enhanced proteomic method, called time-lapse tryptic digestion, in collaboration with Dr. Yu Gao’s lab. With multiple timepoints of the method, we achieved an improved sequence coverage metric, which allows a more thorough survey of the extracellular landscape, with potential to study the post-translational modifications. Furthermore, with temporal aspect of the method, we were able to identify the different segments of proteins released with respect to increased digestion duration. We speculate that the peptides released earlier are more readily accessible to trypsin digestion compared to those released later. Hence, we proposed that these changes in spatial accessibility can identify potential changes in protein folding and organization with respect to SNED1’s presence in the ECM. Next, to investigate how these differences in ECM composition arise, I characterized the ECM proteins secreted by the cells but not yet integrated into the ECM using proteomics. To profile the compositions of secreted proteins, or secretome, without the technical difficulties posed by highly abundant serum proteins in culture media, I optimized a method to collect the secreted proteins without serum proteins. Using this method, I profiled and compared the secretomes of cells lacking or overexpressing SNED1. The preliminary result suggested slight differences in the relative abundances of secreted structural ECM proteins between cells overexpressing SNED1 and their control counterparts. My most recent work was focused on identifying the potential interactors of SNED1 in-situ. With time-lapse tryptic digestion, I identified the SNED1-dependent changes in matrisomes and tryptic accessibilities. However, my preliminary secretomes of the cells lacking or overexpressing SNED1 showed a different set of SNED1-dependent changes. While preliminary, I hypothesized that these discrepancies could be due to SNED1’s possible interaction with other ECM proteins, a lack of which could lead to changes in ECM assembly and deposition. Thus, to test this, I aimed to identify the interactome of SNED1 in-situ. I adapted biotinylation by antibody recognition to the ECM samples, and have optimized the labeling reaction to experimentally identify an ECM interactome for the first time. I propose that the methods I have developed can become the next pipeline of ECM proteomics. The continued work on SNED1 with these methods will shed light on the ECM complexes formed by SNED1 and provide the first step towards deciphering the role of SNED1 in breast cancer metastasis.","abstract_html":"The extracellular matrix (ECM) is a complex meshwork of proteins that provides not only structural support but also biochemical cues to cells. The assembly of the ECM is a dynamic process requiring precise coordination: 1) ECM proteins are produced, post-translationally modified, and then secreted into the extracellular space, 2) once secreted, ECM proteins are organized into protein networks with the aid of the other ECM proteins and ECM receptors on the cell surface, 3) this network of proteins is further maintained and remodeled over time to carry out structural and biochemical roles affecting cell behavior. Thus, a finely tuned balance of the proteins that form and maintain the ECM is essential to health, while an imbalance can cause or accompany physiological and pathological changes, ranging from the wrinkles of the skin to fibrotic diseases and cancer metastases. Despite the significant roles of the ECM in health and disease, studying the ECM had been difficult for a long time due to its biochemically complex nature. However, my mentor, Dr. Alexandra Naba developed a proteomic pipeline tailored to study the protein composition of the ECM, which has since become the method of choice in studying the ECM. Using this method, Dr. Naba identified a novel ECM protein, SNED1, as being only present in the ECM of highly metastatic mammary tumors. Upon this discovery, Dr. Naba knocked down SNED1 in a mammary tumor cell line. When the resulting cells were intravenously injected to mimic the already extravasated tumor cells, the SNED1 knockdown cells did not result in a reduced metastasis. However, when orthotopically injected into mice, SNED1 knockdown cells resulted in fewer metastatic foci compared to the control counterparts, suggesting SNED1 is a protein promoting the invasiveness of the tumor cells. When I started my PhD, not much was known about SNED1. Previous work of the lab on the SNED1 knockout mouse model revealed that SNED1 is essential gene for development. Further work on this aspect by a lab alumna led to identification of SNED1’s involvement in neural crest cell migration. On the other hand, second harmonic generation microscopy revealed that presence of SNED1 altered the ECM organization, but the SNED1-dependent changes in the matrisome was not yet identified. Thus, I hypothesized that SNED1 modulates the ECM architecture by influencing the ECM composition. In this thesis, I focused on answering this biological question and on enhancing proteomic methods to do so, using mouse embryonic fibroblasts derived from a SNED1-knockout mice engineered to overexpress SNED1 or not. To characterize the SNED1-dependent changes in the ECM composition, I used ECM proteomics to compare the compositions of ECMs made by cells overexpressing or not SNED1. To additionally increase the sequence coverage of the ECM proteins, I developed an enhanced proteomic method, called time-lapse tryptic digestion, in collaboration with Dr. Yu Gao’s lab. With multiple timepoints of the method, we achieved an improved sequence coverage metric, which allows a more thorough survey of the extracellular landscape, with potential to study the post-translational modifications. Furthermore, with temporal aspect of the method, we were able to identify the different segments of proteins released with respect to increased digestion duration. We speculate that the peptides released earlier are more readily accessible to trypsin digestion compared to those released later. Hence, we proposed that these changes in spatial accessibility can identify potential changes in protein folding and organization with respect to SNED1’s presence in the ECM. Next, to investigate how these differences in ECM composition arise, I characterized the ECM proteins secreted by the cells but not yet integrated into the ECM using proteomics. To profile the compositions of secreted proteins, or secretome, without the technical difficulties posed by highly abundant serum proteins in culture media, I optimized a method to collect the secreted proteins without serum proteins. Using this method, I profiled and compared the secretomes of cells lacking or overexpressing SNED1. The preliminary result suggested slight differences in the relative abundances of secreted structural ECM proteins between cells overexpressing SNED1 and their control counterparts. My most recent work was focused on identifying the potential interactors of SNED1 in-situ. With time-lapse tryptic digestion, I identified the SNED1-dependent changes in matrisomes and tryptic accessibilities. However, my preliminary secretomes of the cells lacking or overexpressing SNED1 showed a different set of SNED1-dependent changes. While preliminary, I hypothesized that these discrepancies could be due to SNED1’s possible interaction with other ECM proteins, a lack of which could lead to changes in ECM assembly and deposition. Thus, to test this, I aimed to identify the interactome of SNED1 in-situ. I adapted biotinylation by antibody recognition to the ECM samples, and have optimized the labeling reaction to experimentally identify an ECM interactome for the first time. I propose that the methods I have developed can become the next pipeline of ECM proteomics. The continued work on SNED1 with these methods will shed light on the ECM complexes formed by SNED1 and provide the first step towards deciphering the role of SNED1 in breast cancer metastasis.","abstract_has_math":false,"creators":["Fred Lee (13814200)"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2026,"date_issued":"2026-05-01T00:00:00Z","date_published":"2026-05-01T00:00:00Z","updated_at":"2026-07-27T21:33:52Z","subjects":["Biology","Cell"],"languages":[],"rights":["In Copyright","Open Access after 2028-05-01"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.25417/uic.32995250.v1","outbound_label":"DOI","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Fred Lee (13814200)"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2026-05-01T00:00:00Z"]},{"key":"dc:relation","label":"Dc Relation","values":["https://figshare.com/articles/thesis/Enhanced_Proteomic_Methods_to_Decipher_How_the_Novel_Protein_SNED1_Modulates_the_Extracellular_Matrix/32995250"]},{"key":"dc:type","label":"Dc Type","values":["Text","Thesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Biology","Cell"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["In Copyright","Open Access after 2028-05-01"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["10.25417/uic.32995250.v1"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["The extracellular matrix (ECM) is a complex meshwork of proteins that provides not only structural support but also biochemical cues to cells. The assembly of the ECM is a dynamic process requiring precise coordination: 1) ECM proteins are produced, post-translationally modified, and then secreted into the extracellular space, 2) once secreted, ECM proteins are organized into protein networks with the aid of the other ECM proteins and ECM receptors on the cell surface, 3) this network of proteins is further maintained and remodeled over time to carry out structural and biochemical roles affecting cell behavior. Thus, a finely tuned balance of the proteins that form and maintain the ECM is essential to health, while an imbalance can cause or accompany physiological and pathological changes, ranging from the wrinkles of the skin to fibrotic diseases and cancer metastases. Despite the significant roles of the ECM in health and disease, studying the ECM had been difficult for a long time due to its biochemically complex nature. However, my mentor, Dr. Alexandra Naba developed a proteomic pipeline tailored to study the protein composition of the ECM, which has since become the method of choice in studying the ECM. Using this method, Dr. Naba identified a novel ECM protein, SNED1, as being only present in the ECM of highly metastatic mammary tumors. Upon this discovery, Dr. Naba knocked down SNED1 in a mammary tumor cell line. When the resulting cells were intravenously injected to mimic the already extravasated tumor cells, the SNED1 knockdown cells did not result in a reduced metastasis. However, when orthotopically injected into mice, SNED1 knockdown cells resulted in fewer metastatic foci compared to the control counterparts, suggesting SNED1 is a protein promoting the invasiveness of the tumor cells. When I started my PhD, not much was known about SNED1. Previous work of the lab on the SNED1 knockout mouse model revealed that SNED1 is essential gene for development. Further work on this aspect by a lab alumna led to identification of SNED1’s involvement in neural crest cell migration. On the other hand, second harmonic generation microscopy revealed that presence of SNED1 altered the ECM organization, but the SNED1-dependent changes in the matrisome was not yet identified. Thus, I hypothesized that SNED1 modulates the ECM architecture by influencing the ECM composition. In this thesis, I focused on answering this biological question and on enhancing proteomic methods to do so, using mouse embryonic fibroblasts derived from a SNED1-knockout mice engineered to overexpress SNED1 or not. To characterize the SNED1-dependent changes in the ECM composition, I used ECM proteomics to compare the compositions of ECMs made by cells overexpressing or not SNED1. To additionally increase the sequence coverage of the ECM proteins, I developed an enhanced proteomic method, called time-lapse tryptic digestion, in collaboration with Dr. Yu Gao’s lab. With multiple timepoints of the method, we achieved an improved sequence coverage metric, which allows a more thorough survey of the extracellular landscape, with potential to study the post-translational modifications. Furthermore, with temporal aspect of the method, we were able to identify the different segments of proteins released with respect to increased digestion duration. We speculate that the peptides released earlier are more readily accessible to trypsin digestion compared to those released later. Hence, we proposed that these changes in spatial accessibility can identify potential changes in protein folding and organization with respect to SNED1’s presence in the ECM. Next, to investigate how these differences in ECM composition arise, I characterized the ECM proteins secreted by the cells but not yet integrated into the ECM using proteomics. To profile the compositions of secreted proteins, or secretome, without the technical difficulties posed by highly abundant serum proteins in culture media, I optimized a method to collect the secreted proteins without serum proteins. Using this method, I profiled and compared the secretomes of cells lacking or overexpressing SNED1. The preliminary result suggested slight differences in the relative abundances of secreted structural ECM proteins between cells overexpressing SNED1 and their control counterparts. My most recent work was focused on identifying the potential interactors of SNED1 in-situ. With time-lapse tryptic digestion, I identified the SNED1-dependent changes in matrisomes and tryptic accessibilities. However, my preliminary secretomes of the cells lacking or overexpressing SNED1 showed a different set of SNED1-dependent changes. While preliminary, I hypothesized that these discrepancies could be due to SNED1’s possible interaction with other ECM proteins, a lack of which could lead to changes in ECM assembly and deposition. Thus, to test this, I aimed to identify the interactome of SNED1 in-situ. I adapted biotinylation by antibody recognition to the ECM samples, and have optimized the labeling reaction to experimentally identify an ECM interactome for the first time. I propose that the methods I have developed can become the next pipeline of ECM proteomics. The continued work on SNED1 with these methods will shed light on the ECM complexes formed by SNED1 and provide the first step towards deciphering the role of SNED1 in breast cancer metastasis."]},{"key":"dc:title","label":"Title","values":["Enhanced Proteomic Methods to Decipher How the Novel Protein SNED1 Modulates the Extracellular Matrix"]}]}],"canonical_facts":{"dc:creator":["Fred Lee (13814200)"],"dc:date":["2026-05-01T00:00:00Z"],"dc:description":["The extracellular matrix (ECM) is a complex meshwork of proteins that provides not only structural support but also biochemical cues to cells. The assembly of the ECM is a dynamic process requiring precise coordination: 1) ECM proteins are produced, post-translationally modified, and then secreted into the extracellular space, 2) once secreted, ECM proteins are organized into protein networks with the aid of the other ECM proteins and ECM receptors on the cell surface, 3) this network of proteins is further maintained and remodeled over time to carry out structural and biochemical roles affecting cell behavior. Thus, a finely tuned balance of the proteins that form and maintain the ECM is essential to health, while an imbalance can cause or accompany physiological and pathological changes, ranging from the wrinkles of the skin to fibrotic diseases and cancer metastases. Despite the significant roles of the ECM in health and disease, studying the ECM had been difficult for a long time due to its biochemically complex nature. However, my mentor, Dr. Alexandra Naba developed a proteomic pipeline tailored to study the protein composition of the ECM, which has since become the method of choice in studying the ECM. Using this method, Dr. Naba identified a novel ECM protein, SNED1, as being only present in the ECM of highly metastatic mammary tumors. Upon this discovery, Dr. Naba knocked down SNED1 in a mammary tumor cell line. When the resulting cells were intravenously injected to mimic the already extravasated tumor cells, the SNED1 knockdown cells did not result in a reduced metastasis. However, when orthotopically injected into mice, SNED1 knockdown cells resulted in fewer metastatic foci compared to the control counterparts, suggesting SNED1 is a protein promoting the invasiveness of the tumor cells. When I started my PhD, not much was known about SNED1. Previous work of the lab on the SNED1 knockout mouse model revealed that SNED1 is essential gene for development. Further work on this aspect by a lab alumna led to identification of SNED1’s involvement in neural crest cell migration. On the other hand, second harmonic generation microscopy revealed that presence of SNED1 altered the ECM organization, but the SNED1-dependent changes in the matrisome was not yet identified. Thus, I hypothesized that SNED1 modulates the ECM architecture by influencing the ECM composition. In this thesis, I focused on answering this biological question and on enhancing proteomic methods to do so, using mouse embryonic fibroblasts derived from a SNED1-knockout mice engineered to overexpress SNED1 or not. To characterize the SNED1-dependent changes in the ECM composition, I used ECM proteomics to compare the compositions of ECMs made by cells overexpressing or not SNED1. To additionally increase the sequence coverage of the ECM proteins, I developed an enhanced proteomic method, called time-lapse tryptic digestion, in collaboration with Dr. Yu Gao’s lab. With multiple timepoints of the method, we achieved an improved sequence coverage metric, which allows a more thorough survey of the extracellular landscape, with potential to study the post-translational modifications. Furthermore, with temporal aspect of the method, we were able to identify the different segments of proteins released with respect to increased digestion duration. We speculate that the peptides released earlier are more readily accessible to trypsin digestion compared to those released later. Hence, we proposed that these changes in spatial accessibility can identify potential changes in protein folding and organization with respect to SNED1’s presence in the ECM. Next, to investigate how these differences in ECM composition arise, I characterized the ECM proteins secreted by the cells but not yet integrated into the ECM using proteomics. To profile the compositions of secreted proteins, or secretome, without the technical difficulties posed by highly abundant serum proteins in culture media, I optimized a method to collect the secreted proteins without serum proteins. Using this method, I profiled and compared the secretomes of cells lacking or overexpressing SNED1. The preliminary result suggested slight differences in the relative abundances of secreted structural ECM proteins between cells overexpressing SNED1 and their control counterparts. My most recent work was focused on identifying the potential interactors of SNED1 in-situ. With time-lapse tryptic digestion, I identified the SNED1-dependent changes in matrisomes and tryptic accessibilities. However, my preliminary secretomes of the cells lacking or overexpressing SNED1 showed a different set of SNED1-dependent changes. While preliminary, I hypothesized that these discrepancies could be due to SNED1’s possible interaction with other ECM proteins, a lack of which could lead to changes in ECM assembly and deposition. Thus, to test this, I aimed to identify the interactome of SNED1 in-situ. I adapted biotinylation by antibody recognition to the ECM samples, and have optimized the labeling reaction to experimentally identify an ECM interactome for the first time. I propose that the methods I have developed can become the next pipeline of ECM proteomics. The continued work on SNED1 with these methods will shed light on the ECM complexes formed by SNED1 and provide the first step towards deciphering the role of SNED1 in breast cancer metastasis."],"dc:identifier":["10.25417/uic.32995250.v1"],"dc:relation":["https://figshare.com/articles/thesis/Enhanced_Proteomic_Methods_to_Decipher_How_the_Novel_Protein_SNED1_Modulates_the_Extracellular_Matrix/32995250"],"dc:rights":["In Copyright","Open Access after 2028-05-01"],"dc:subject":["Biology","Cell"],"dc:title":["Enhanced Proteomic Methods to Decipher How the Novel Protein SNED1 Modulates the Extracellular Matrix"],"dc:type":["Text","Thesis"]},"updated_at":"2026-07-27T21:33:52Z"}