{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T14:28:14Z","timestamp":1788532094451,"version":"build-2803163510"},"reference-count":30,"publisher":"American Chemical Society (ACS)","issue":"9","license":[{"start":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T00:00:00Z","timestamp":1787097600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Profiling of newly synthesized proteins (NSPs) provides access to dynamic changes in protein production that accompany acute cellular responses. Bioorthogonal noncanonical amino acid tagging (BONCAT)-based approaches enable selective labeling of NSPs; however, their broader application remains constrained by labor-intensive enrichment workflows and limited sensitivity for direct peptide-level analysis. Here, we developed a workflow termed \u201cPhos-tag Click Tip\u201d by integrating a phosphorylated variant of bicyclononyne (pBCN) with Phos-tag affinity purification to selectively capture azidohomoalanine (AHA)-labeled peptides for newly synthesized proteome analysis (NSProteomics). This approach overcomes key limitations of conventional proteomics and BONCAT-based strategies by enabling efficient enrichment and sensitive detection of NSP-derived peptides. Using this workflow, we performed comprehensive NSP profiling of host cells during influenza A virus infection. We identified dynamic changes in distinct NSP profiles associated with viral replication, host restriction, and immune responses, many of which were not readily detected with conventional whole-cell- or phospho-proteomic analyses. Overall, the Phos-tag Click Tip workflow provides a complementary approach for stimulus-responsive NSP profiling, offering functionally relevant insights into host\u2013virus interactions and cellular response mechanisms.<\/jats:p>","DOI":"10.1021\/acs.jproteome.6c00391","type":"journal-article","created":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T22:01:17Z","timestamp":1787176877000},"page":"4888-4902","source":"Crossref","is-referenced-by-count":0,"title":["Selective Enrichment\nof Newly Synthesized Proteins\nUsing Phos-Tag Click Tip Enables Nascent Proteome Analysis in Influenza\nA Virus Infection"],"prefix":"10.1021","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4612-4602","authenticated-orcid":true,"given":"Yayoi","family":"Kimura","sequence":"first","affiliation":[{"name":"Yokohama City University , , ,","place":["Yokohama, Japan, 236-0004"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomoko","family":"Akiyama","sequence":"additional","affiliation":[{"name":"Yokohama City University , , ,","place":["Yokohama, Japan, 236-0004"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tatsuhiro","family":"Kobashi","sequence":"additional","affiliation":[{"name":"NARD Institute, Ltd , ,","place":["Kobe, Japan, 650-0047"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maiko","family":"Hosotani","sequence":"additional","affiliation":[{"name":"Yokohama City University , , ,","place":["Yokohama, Japan, 236-0004"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoko","family":"Ino","sequence":"additional","affiliation":[{"name":"Yokohama City University , , ,","place":["Yokohama, Japan, 236-0004"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jihye","family":"Shin","sequence":"additional","affiliation":[{"name":"Yokohama City University , , ,","place":["Yokohama, Japan, 236-0004"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naotoshi","family":"Nakamura","sequence":"additional","affiliation":[{"name":"Yokohama City University , , ,","place":["Yokohama, Japan, 236-0027"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eiji","family":"Kinoshita","sequence":"additional","affiliation":[{"name":"Hiroshima Bunkyo University , , ,","place":["Hiroshima, Japan, 731-0295"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kei","family":"Miyakawa","sequence":"additional","affiliation":[{"name":"Japan Institute for Health Security , , ,","place":["Tokyo, Japan, 208-0011"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Akihide","family":"Ryo","sequence":"additional","affiliation":[{"name":"Japan Institute for Health Security , , ,","place":["Tokyo, Japan, 208-0011"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,19]]},"reference":[{"issue":"3","key":"2026090409132261000_cit1","doi-asserted-by":"publisher","first-page":"532","DOI":"10.1038\/nprot.2007.52","article-title":"Labeling, detection and identification of newly synthesized\nproteomes with bioorthogonal non-canonical amino-acid tagging","volume":"2","author":"Dieterich","year":"2007","journal-title":"Nat. Protoc."},{"issue":"7","key":"2026090409132261000_cit2","doi-asserted-by":"publisher","first-page":"545","DOI":"10.1080\/14789450.2018.1500902","article-title":"Proteomics and pulse azidohomoalanine\nlabeling of newly synthesized proteins: what are the potential applications?","volume":"15","author":"Ma","year":"2018","journal-title":"Expert Rev. Proteomics"},{"issue":"9","key":"2026090409132261000_cit3","doi-asserted-by":"publisher","first-page":"3966","DOI":"10.1021\/pr5002862","article-title":"Direct detection\nof biotinylated\nproteins by mass spectrometry","volume":"13","author":"Schiapparelli","year":"2014","journal-title":"J. Proteome Res."},{"issue":"10","key":"2026090409132261000_cit4","doi-asserted-by":"publisher","first-page":"3651","DOI":"10.1021\/jacs.6b10418","article-title":"Architecture mapping\nof the inner mitochondrial membrane proteome by chemical tools in\nlive cells","volume":"139","author":"Lee","year":"2017","journal-title":"J. Am. Chem. Soc."},{"issue":"12","key":"2026090409132261000_cit5","doi-asserted-by":"publisher","first-page":"1167","DOI":"10.1038\/nmeth.4465","article-title":"Antibodies\nto biotin\nenable large-scale detection of biotinylation sites on proteins","volume":"14","author":"Udeshi","year":"2017","journal-title":"Nat. Methods"},{"issue":"12","key":"2026090409132261000_cit6","doi-asserted-by":"publisher","first-page":"5487","DOI":"10.1021\/acs.jproteome.4c00616","article-title":"3rd. Acid\ncleavable biotin-alkyne improves sensitivity for direct detection\nof biotin labeled peptides in biorthogonal noncanonical amino acid\ntagging analysis","volume":"23","author":"McClatchy","year":"2024","journal-title":"J. Proteome Res."},{"issue":"1","key":"2026090409132261000_cit7","doi-asserted-by":"publisher","first-page":"110","DOI":"10.1016\/j.cbpa.2012.11.007","article-title":"Chemical proteomics: ligation and\ncleavage of protein modifications","volume":"17","author":"Rudolf","year":"2013","journal-title":"Curr. Opin.\nChem. Biol."},{"issue":"11","key":"2026090409132261000_cit8","doi-asserted-by":"publisher","first-page":"1212","DOI":"10.1016\/j.chembiol.2010.09.012","article-title":"Comparative\nanalysis of cleavable azobenzene-based affinity tags for bioorthogonal\nchemical proteomics","volume":"17","author":"Yang","year":"2010","journal-title":"Chem. Biol."},{"issue":"2","key":"2026090409132261000_cit9","first-page":"628","article-title":"Peptide- and protein-level\ncombined strategy for analyzing newly\nsynthesized proteins by integrating tandem orthogonal proteolysis\nwith cleavable bioorthogonal tagging","volume":"95","author":"Meng","year":"2023","journal-title":"Anal.\nChem."},{"issue":"1","key":"2026090409132261000_cit10","doi-asserted-by":"publisher","first-page":"3244","DOI":"10.1038\/s41467-020-17010-0","article-title":"Fishing for newly\nsynthesized proteins with phosphonate-handles","volume":"11","author":"Kleinpenning","year":"2020","journal-title":"Nat. Commun."},{"issue":"8","key":"2026090409132261000_cit11","doi-asserted-by":"publisher","first-page":"1896","DOI":"10.1038\/nprot.2007.261","article-title":"Protocol for\nmicro-purification,\nenrichment, pre-fractionation and storage of peptides for proteomics\nusing StageTips","volume":"2","author":"Rappsilber","year":"2007","journal-title":"Nat. Protoc."},{"issue":"7","key":"2026090409132261000_cit12","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.202100216","article-title":"Evaluation of four phosphopeptide enrichment strategies\nfor mass spectrometry-based proteomic analysis","volume":"22","author":"Ino","year":"2022","journal-title":"Proteomics"},{"issue":"1","key":"2026090409132261000_cit13","doi-asserted-by":"publisher","first-page":"1523","DOI":"10.1038\/s41467-019-09234-6","article-title":"Metascape provides\na biologist-oriented resource for the analysis of systems-level datasets","volume":"10","author":"Zhou","year":"2019","journal-title":"Nat. Commun."},{"issue":"1","key":"2026090409132261000_cit14","doi-asserted-by":"publisher","first-page":"156","DOI":"10.1016\/j.jviromet.2010.10.018","article-title":"One-step real-time\nreverse transcription-PCR\nassays for detecting and subtyping pandemic influenza A\/H1N1 2009,\nseasonal influenza A\/H1N1, and seasonal influenza A\/H3N2 viruses","volume":"171","author":"Nakauchi","year":"2011","journal-title":"J. Virol. Methods"},{"issue":"4","key":"2026090409132261000_cit15","doi-asserted-by":"publisher","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis\nof relative gene expression data using real-time\nquantitative PCR and the 2(-Delta Delta C(T)) Method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"issue":"46","key":"2026090409132261000_cit16","doi-asserted-by":"publisher","first-page":"15046","DOI":"10.1021\/ja044996f","article-title":"A strain-promoted [3 + 2] azide-alkyne\ncycloaddition for covalent modification of biomolecules in living\nsystems","volume":"126","author":"Agard","year":"2004","journal-title":"J. Am. Chem. Soc."},{"issue":"2","key":"2026090409132261000_cit17","doi-asserted-by":"publisher","first-page":"219","DOI":"10.1002\/rcm.4377","article-title":"Enrichment and analysis of phosphopeptides under different\nexperimental conditions using titanium dioxide affinity chromatography\nand mass spectrometry","volume":"24","author":"Aryal","year":"2010","journal-title":"Rapid Commun. Mass Spectrom."},{"issue":"12","key":"2026090409132261000_cit18","doi-asserted-by":"publisher","first-page":"4666","DOI":"10.1021\/ac0618730","article-title":"Optimizing TiO2-based\nphosphopeptide enrichment for\nautomated multidimensional liquid chromatography coupled to tandem\nmass spectrometry","volume":"79","author":"Cantin","year":"2007","journal-title":"Anal. Chem."},{"issue":"6","key":"2026090409132261000_cit19","doi-asserted-by":"publisher","first-page":"1103","DOI":"10.1074\/mcp.T600060-MCP200","article-title":"Phosphopeptide enrichment\nby aliphatic hydroxy acid-modified\nmetal oxide chromatography for nano-LC-MS\/MS in proteomics applications","volume":"6","author":"Sugiyama","year":"2007","journal-title":"Mol. Cell. Proteomics"},{"issue":"8","key":"2026090409132261000_cit20","doi-asserted-by":"publisher","first-page":"1189","DOI":"10.1039\/b400269e","article-title":"Recognition\nof phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II)\ncomplex","author":"Kinoshita","year":"2004","journal-title":"Dalton Trans."},{"issue":"6","key":"2026090409132261000_cit21","doi-asserted-by":"publisher","first-page":"601","DOI":"10.1016\/j.bbapap.2014.10.004","article-title":"Advances in Phos-tag-based methodologies\nfor separation and detection of the phosphoproteome","volume":"1854","author":"Kinoshita","year":"2015","journal-title":"Biochim. Biophys. Acta Gen. Subj."},{"issue":"2","key":"2026090409132261000_cit22","doi-asserted-by":"publisher","first-page":"155","DOI":"10.1002\/jssc.200401833","article-title":"Novel immobilized zinc(II) affinity\nchromatography for phosphopeptides\nand phosphorylated proteins","volume":"28","author":"Kinoshita","year":"2005","journal-title":"J. Sep. Sci."},{"issue":"24","key":"2026090409132261000_cit23","doi-asserted-by":"publisher","first-page":"5525","DOI":"10.1002\/pmic.200900341","article-title":"Improved method\nof phosphopeptides enrichment using\nbiphasic phosphate-binding tag\/C18 tip for versatile analysis of phosphorylation\ndynamics","volume":"9","author":"Nabetani","year":"2009","journal-title":"Proteomics"},{"key":"2026090409132261000_cit24","doi-asserted-by":"publisher","first-page":"828665","DOI":"10.3389\/fimmu.2022.828665","article-title":"Modulation of mRNA\n3\u2032-end\nprocessing and transcription termination in virus-infected cells","volume":"13","author":"Vijayakumar","year":"2022","journal-title":"Front. Immunol."},{"issue":"36","key":"2026090409132261000_cit25","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2024681118","article-title":"Influenza A viruses balance ER stress with host protein\nsynthesis shutoff","volume":"118","author":"Mazel-Sanchez","year":"2021","journal-title":"Proc. Natl. Acad. Sci. U.\nS. A."},{"issue":"1","key":"2026090409132261000_cit26","doi-asserted-by":"publisher","first-page":"6030","DOI":"10.1038\/s41467-023-41442-z","article-title":"Proteomic and genetic\nanalyses of influenza A viruses identify pan-viral host targets","volume":"14","author":"Haas","year":"2023","journal-title":"Nat. Commun."},{"issue":"24","key":"2026090409132261000_cit27","doi-asserted-by":"publisher","first-page":"17632","DOI":"10.1074\/jbc.M611237200","article-title":"Mammalian\nSec16\/p250 plays a role in membrane traffic from the endoplasmic reticulum","volume":"282","author":"Iinuma","year":"2007","journal-title":"J. Biol. Chem."},{"key":"2026090409132261000_cit28","doi-asserted-by":"publisher","first-page":"39887","DOI":"10.1038\/srep39887","article-title":"Sec16A\nis critical for both conventional and unconventional secretion of\nCFTR","volume":"7","author":"Piao","year":"2017","journal-title":"Sci. Rep"},{"issue":"31","key":"2026090409132261000_cit29","doi-asserted-by":"publisher","first-page":"12746","DOI":"10.1073\/pnas.1103283108","article-title":"Sec16B is involved\nin the endoplasmic reticulum export of the peroxisomal membrane biogenesis\nfactor peroxin 16 (Pex16) in mammalian cells","volume":"108","author":"Yonekawa","year":"2011","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"D1","key":"2026090409132261000_cit30","doi-asserted-by":"publisher","first-page":"D462","DOI":"10.1093\/nar\/gkae1032","article-title":"jPOST\nenvironment accelerates\nthe reuse and reanalysis of public proteome mass spectrometry data","volume":"53","author":"Okuda","year":"2025","journal-title":"Nucleic Acids Res."}],"container-title":["Journal of Proteome Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jproteome.6c00391","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jprobs\/article-pdf\/25\/9\/4888\/66884976\/acs.jproteome.6c00391.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jprobs\/article-pdf\/25\/9\/4888\/66884976\/acs.jproteome.6c00391.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T14:05:34Z","timestamp":1788530734000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jprobs\/article\/25\/9\/4888\/5277077\/Selective-Enrichment-of-Newly-Synthesized-Proteins"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,19]]},"references-count":30,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,8,19]]},"published-print":{"date-parts":[[2026,9,4]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jproteome.6c00391","relation":{},"ISSN":["1535-3893","1535-3907"],"issn-type":[{"value":"1535-3893","type":"print"},{"value":"1535-3907","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,19]]}}}