{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,28]],"date-time":"2026-01-28T12:57:17Z","timestamp":1769605037694,"version":"3.49.0"},"reference-count":28,"publisher":"Wiley","issue":"16","license":[{"start":{"date-parts":[[2025,8,5]],"date-time":"2025-08-05T00:00:00Z","timestamp":1754352000000},"content-version":"vor","delay-in-days":4,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/100000054","name":"National Cancer Institute","doi-asserted-by":"publisher","award":["U01CA271410"],"award-info":[{"award-number":["U01CA271410"]}],"id":[{"id":"10.13039\/100000054","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000054","name":"National Cancer Institute","doi-asserted-by":"publisher","award":["P30CA15083"],"award-info":[{"award-number":["P30CA15083"]}],"id":[{"id":"10.13039\/100000054","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Proteomics"],"published-print":{"date-parts":[[2025,8]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:label\/>\n                    <jats:p>T\u2010cell receptor (TCR) signaling plays a crucial role in various biological processes and is usually studied using global mass spectrometry\u2010based phosphoproteomic studies. Despite advancements in targeted mass spectrometry\u2010based assays for protein quantification, their application in studying signaling processes, for example, reproducible measurements of post\u2010translational modifications (PTMs) such as phosphorylation, remains limited. Tyrosine phosphorylation is critical for many signaling pathways but presents challenges due to the low abundance of phosphotyrosine\u2010containing peptides. Conventional untargeted methods often encounter data gaps when analyzing large sample sets, particularly for low\u2010abundance peptides. To address this issue, a targeted proteomics method called \u201cSureQuant\u201d was employed, which relies on triggered data acquisition with heavy isotope\u2010labeled peptides. This method has been shown to provide sensitive and reproducible quantification of low\u2010abundance peptides. Here we describe the development of a SureQuant\u2010based method to quantify phosphotyrosine peptides that are involved in the TCR signaling pathway. To monitor the change in phosphotyrosine signals upon activation, the T\u2010cells were stimulated with anti\u2010CD3\/CD28 antibodies. We successfully quantified changes in important phosphotyrosine peptides in primary T\u2010cells upon stimulation with anti\u2010CD3\/CD28 antibodies. This study showcases the ability of the SureQuant approach to accurately quantify low\u2010abundance phosphotyrosine peptides, highlighting its broader potential to study a diverse set of PTMs in physiological or clinical settings.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Summary<\/jats:title>\n                    <jats:p>\n                      <jats:list list-type=\"bullet\">\n                        <jats:list-item>\n                          <jats:p>T\u2010cell receptor (TCR) signaling plays a fundamental role in immune responses, regulating T\u2010cell activation, differentiation, and function. While tyrosine phosphorylation is a key regulatory mechanism in this pathway, the low abundance of phosphotyrosine peptides presents a major challenge for their detection and quantification in complex biological samples. By employing the SureQuant targeted mass spectrometry approach, we achieved highly sensitive and reproducible quantification of key phosphotyrosine sites involved in T\u2010cell activation.<\/jats:p>\n                        <\/jats:list-item>\n                        <jats:list-item>\n                          <jats:p>This study provides a systematic view of TCR signaling dynamics, revealing distinct phosphorylation patterns across different activation timepoints. Our findings demonstrate the effectiveness of SureQuant in quantifying low\u2010abundance, post\u2010translationally modified peptides, offering a valuable tool for studying signaling pathways with greater precision.<\/jats:p>\n                        <\/jats:list-item>\n                        <jats:list-item>\n                          <jats:p>Additionally, this methodological framework can be extended to investigate other signaling networks, immune cell functions, and disease\u2010associated phosphotyrosine modifications.<\/jats:p>\n                        <\/jats:list-item>\n                      <\/jats:list>\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/pmic.70023","type":"journal-article","created":{"date-parts":[[2025,8,5]],"date-time":"2025-08-05T08:05:46Z","timestamp":1754381146000},"page":"40-47","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Targeted Quantitation of Phosphotyrosine\u2010Containing Proteins in T\u2010Cell Receptor Signaling Using a SureQuant\u2010Based Mass Spectrometry Approach"],"prefix":"10.1002","volume":"25","author":[{"given":"Firdous A.","family":"Bhat","sequence":"first","affiliation":[{"name":"Department of Laboratory Medicine and Pathology Mayo Clinic  Rochester Minnesota USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Husheng","family":"Ding","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology Mayo Clinic  Rochester Minnesota USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dong\u2010Gi","family":"Mun","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology Mayo Clinic  Rochester Minnesota USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jane A.","family":"Peterson","sequence":"additional","affiliation":[{"name":"Proteomics Core Mayo Clinic  Rochester Minnesota USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mary Cristine","family":"Charlesworth","sequence":"additional","affiliation":[{"name":"Proteomics Core Mayo Clinic  Rochester Minnesota USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Richard K.","family":"Kandasamy","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology Mayo Clinic  Rochester Minnesota USA"},{"name":"Department of Quantitative Health Sciences Mayo Clinic  Rochester Minnesota USA"},{"name":"Department of Immunology Mayo Clinic  Rochester Minnesota USA"},{"name":"Center For Individualized Medicine Mayo Clinic  Rochester Minnesota USA"},{"name":"Manipal Academy of Higher Education  Manipal Karnataka India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9943-6127","authenticated-orcid":false,"given":"Akhilesh","family":"Pandey","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology Mayo Clinic  Rochester Minnesota USA"},{"name":"Center For Individualized Medicine Mayo Clinic  Rochester Minnesota USA"},{"name":"Manipal Academy of Higher Education  Manipal Karnataka India"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2025,8,5]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1124619"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.201500449"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2015"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/msb.2008.61"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1074\/mcp.O112.020131"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1074\/mcp.O114.043968"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-20-3804"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.immunol.021908.132706"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1038\/ni.2941"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M110.201236"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jproteome.6b00576"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1074\/mcp.M800307-MCP200"},{"key":"e_1_2_9_14_1","first-page":"ra49","article-title":"The Catalytic Activity of the Kinase ZAP\u201070 Mediates Basal Signaling and Negative Feedback of the T Cell Receptor Pathway","volume":"8","author":"Goodfellow H. 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