JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST003525/files/240530QE_Total_Mock_2.rawhttps://storage.jpostdb.org/JPST003525/files/240529QE_phospho_Mock_2.rawhttps://storage.jpostdb.org/JPST003525/files/240524HF_Boncat_IAV_1.rawhttps://storage.jpostdb.org/JPST003525/files/240524HF_Boncat_IAV_3.rawhttps://storage.jpostdb.org/JPST003525/files/240530QE_Total_IAV_2.rawhttps://storage.jpostdb.org/JPST003525/files/240524HF_Boncat_Mock_2.rawhttps://storage.jpostdb.org/JPST003525/files/240529QE_phospho_IAV_2.rawhttps://storage.jpostdb.org/JPST003525/files/240530QE_Total_Mock_1.rawhttps://storage.jpostdb.org/JPST003525/files/240530QE_Total_Mock_3.rawhttps://storage.jpostdb.org/JPST003525/files/240529QE_phospho_Mock_1.rawhttps://storage.jpostdb.org/JPST003525/files/240529QE_phospho_Mock_3.rawhttps://storage.jpostdb.org/JPST003525/files/240530QE_Total_IAV_1.rawhttps://storage.jpostdb.org/JPST003525/files/240524HF_Boncat_IAV_2.rawhttps://storage.jpostdb.org/JPST003525/files/240529QE_phospho_IAV_1.rawhttps://storage.jpostdb.org/JPST003525/files/240524HF_Boncat_Mock_1.rawhttps://storage.jpostdb.org/JPST003525/files/240524HF_Boncat_Mock_3.rawhttps://storage.jpostdb.org/JPST003525/files/240529QE_phospho_IAV_3.rawhttps://storage.jpostdb.org/JPST003525/files/240530QE_Total_IAV_3.rawhttps://storage.jpostdb.org/JPST003525/files/20240529%20Phospho_Mock%20vs%20IAV_limit500.mgfhttps://storage.jpostdb.org/JPST003525/files/20240530_Total_Mock%20vs%20IAV.mgfprimaryOK200ProteomicsYayoi KimuraHomo Sapiens (human)https://repository.jpostdb.org/entry/JPST003525Yokohama City UniversityjPOSTProfiling 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 "Phos-tag Click Tip" 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-virus interactions and cellular response mechanisms.Selective Enrichment of Newly Synthesized Proteins Using Phos-Tag Click Tip Enables Nascent Proteome Analysis in Influenza A Virus Infection.Kimura Yayoi Y, Akiyama Tomoko T, Kobashi Tatsuhiro T, Hosotani Maiko M, Ino Yoko Y, Shin Jihye J, Nakamura Naotoshi N, Kinoshita Eiji E, Miyakawa Kei K, Ryo Akihide AfalseSelective Enrichment of Newly Synthesized Proteins using Phos-tag Click Tip Enables Nascent Proteome Analysis in Influenza A Virus InfectionBioorthogonal non-canonical amino acid tagging (BONCAT)-based approaches enable selective labeling of newly synthesized proteins (NSPs). We developed a workflow termed the “Phos-tag Click Tip” by integrating a phosphorylated variant of bicyclononyne (pBCN) with Phos-tag affinity purification to selectively capture azidohomoalanine (AHA)-labeled peptides. Applying this workflow, we performed comprehensive NSP profiling of host cells during influenza A virus (IAV) infection. We identified dynamic changes in NSP profiles associated with viral replication, host restriction, and immune responses, many of which were not readily detected by conventional whole-cell or phosphoproteomic analyses. Wed Sep 09 00:00:00 BST 2026PXD059239960642695899