biostudies-literatureKundu SNational Institute of General Medical SciencesNIGMS NIH HHS9576-9584https://www.ebi.ac.uk/biostudies/studies/S-EPMC11299218biostudies-literatureUnknown96(23)GPI-anchored proteins (GPI-APs) are ubiquitous and essential but exist in low abundances on the cell surface, making their analysis and investigation especially challenging. To tackle the problem, a new method to detect and study GPI-APs based upon GPI metabolic engineering and DNA-facilitated fluorescence signal amplification was developed. In this context, cell surface GPI-APs were metabolically engineered using azido-inositol derivatives to introduce an azido group. This allowed GPI-AP coupling with alkyne-functionalized multifluorophore DNA assemblies generated by hybridization chain reaction (HCR). It was demonstrated that this approach could significantly improve the detection limit and sensitivity of GPI-APs, thereby enabling various biological studies, including the investigation oAnalytical chemistrySensitive Method To Analyze Cell Surface GPI-Anchored Proteins Using DNA Hybridization Chain Reaction-Mediated Signal Amplification.PMC11299218R35 GM131686GM131686Guo JGuo ZCraig KCGupta PJaiswal MKundu SfalseSensitive Method To Analyze Cell Surface GPI-Anchored Proteins Using DNA Hybridization Chain Reaction-Mediated Signal Amplification.GPI-anchored proteins (GPI-APs) are ubiquitous and essential but exist in low abundances on the cell surface, making their analysis and investigation especially challenging. To tackle the problem, a new method to detect and study GPI-APs based upon GPI metabolic engineering and DNA-facilitated fluorescence signal amplification was developed. In this context, cell surface GPI-APs were metabolically engineered using azido-inositol derivatives to introduce an azido group. This allowed GPI-AP coupling with alkyne-functionalized multifluorophore DNA assemblies generated by hybridization chain reaction (HCR). It was demonstrated that this approach could significantly improve the detection limit and sensitivity of GPI-APs, thereby enabling various biological studies, including the investigation o2024-01-01T00:00:00Z2024 Jun2025-04-27T00:14:46.223Z2025-04-06T17:47:18.112ZS-EPMC112992183880892310.1021/acs.analchem.4c01116