JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST004735/files/Data%20list_SALSA-Peptide.xlsxhttps://storage.jpostdb.org/JPST004735/files/PD2.4-Byonic3.10%20(hPlasma_O-Glycan).ziphttps://storage.jpostdb.org/JPST004735/files/PD2.4-Byonic3.8%20(hAGP_N-Glycan).ziphttps://storage.jpostdb.org/JPST004735/files/Q%20Exactive%20(hPlasma_O-Glycan).ziphttps://storage.jpostdb.org/JPST004735/files/PD2.4-Byonic3.8%20(hFET_N+O-Glycan).ziphttps://storage.jpostdb.org/JPST004735/files/PD3.2-Byonic6.0%20(hAGP_N-Glycan).ziphttps://storage.jpostdb.org/JPST004735/files/Q%20Exactive%20(hFET_N+O-Glycan).ziphttps://storage.jpostdb.org/JPST004735/files/Q%20Exactive%20(hAGP_N-Glycan).ziphttps://storage.jpostdb.org/JPST004735/files/Q%20Exactive%20(SGP).ziphttps://storage.jpostdb.org/JPST004735/files/PD2.4-Byonic3.10%20(hPlasma_N-Glycan).ziphttps://storage.jpostdb.org/JPST004735/files/PD3.2-Byonic6.0%20(hFET_N+O-Glycan).ziphttps://storage.jpostdb.org/JPST004735/files/Q%20Exactive%20(hPlasma_N-Glycan).zipprimaryOK200ProteomicsYuri MiuraHomo Sapiens (human)https://repository.jpostdb.org/entry/JPST004735Tokyo Metropolitan Institute for Geriatrics and GerontologyjPOSTSialic acids are attached to glycoproteins via α2,3- or α2,6-linkages and play important roles in disease pathogenesis. Therefore, accurate discrimination and quantification of these linkage isomers are essential. Although several chemical derivatization strategies to discriminate the isomers by mass differences have been developed for the analysis of released <i>N</i>-glycans, sialyl linkage-specific derivatization of intact <i>N</i>- and/or <i>O</i>-glycopeptides remains challenging because of the diverse reactive functional groups within peptides. Despite previous efforts, a general strategy enabling <i>N</i>- and <i>O</i>-glycoproteomics with reliable α2,3/α2,6 discrimination has not been established. In this report, we present a workflow for identifying and quantifying sialylated <i>N</i>- and <i>O</i>-glycopeptides that integrates dimethyl labeling for amino-group blocking, sialic acid linkage-specific alkylamidation, and label-free quantification. The method was validated using two standard α2,3- and α2,6-sialylglycopeptides and purified <i>N</i>- and <i>O</i>-glycoproteins, including human fetuin and the alpha-1-acid glycoprotein, and subsequently applied to human pooled plasma. This approach enabled the identification of 831 sialylated <i>N</i>-glycopeptides from 79 proteins and 75 sialylated <i>O</i>-glycopeptides from 29 proteins in human plasma. The proposed platform provides a powerful and versatile strategy for discriminating α2,3- and α2,6-sialyl linkages at the glycopeptide level, with broad potential applications in <i>N</i>- and <i>O</i>-glycoproteomics for disease diagnosis, drug discovery, and innovative therapy development.Sialic Acid Linkage-Specific Alkylamidation-Based Method for &lt;i&gt;N&lt;/i&gt;- and &lt;i&gt;O&lt;/i&gt;-Glycoproteomic Profiling of Human Plasma.Tsumoto Hiroki H, Nishikaze Takashi T, Sekiya Sadanori S, Umezawa Keitaro K, Kawakami Kyojiro K, Masui Yukie Y, Ikebe Kazunori K, Kamide Kei K, Hirata Takumi T, Gondo Yasuyuki Y, Iwamoto Shinichi S, Tanaka Koichi K, Miura Yuri YfalseSialic Acid Linkage-Specific Alkylamidation-Based Method for Comprehensive N- and O-Glycoproteomic Profiling of Human PlasmaSialic acids are attached to glycoproteins via alpha2,3- or alpha2,6-linkages and play important roles in disease pathogenesis. Sialyl linkage-specific derivatization of intact N- and/or O-glycopeptides remains challenging because of the diverse reactive functional groups within peptides. In this report, we present a comprehensive workflow for identifying and quantifying sialylated N- and O-glycopeptides that integrates dimethyl labeling for amino-group blocking, sialic acid linkage-specific alkylamidation, and label-free quantification.Wed Jun 10 00:00:00 BST 2026PXD080569960642421262