Detecting Proteins Glycosylation by a Homogeneous Reac-tion System with Zwitterionic Gold Nanoclusters

Homogeneous gold nanoclusters (Au NCs) have been widely utilized in drug delivery, chemical sensing, bioassays and bio-labeling due to their unique physical and chemical properties. However, few attentions have been paid on their application in detecting protein post-translational modifications. Herein, we developed a homogeneous reaction system with water-soluble zwitterionic Au NCs to capture glycopeptides from the complex biological samples for the first time. The unique characteristics of Au NCs, such as the molecular-like property, the excellent homogeneity in aqueous solution and organic solvent responsive precipitation, the easy preparation in only 4.5 hours, contribute to the high efficiency and high through-put for capturing the targeted glycopeptides. Briefly, the Au NCs and tryptic protein samples were incubated together in a homogeneous aqueous solution and then the glycopeptides were collected by co-precipitation with zwitterionic Au NCs dur-ing gradually dropping addition of acetonitrile. Compared with the conventional heterogeneous system with solid-state ad-sorbents, the number of characterized glycosylation sites was improved 35%. Finally, the MS detection limitation as low as 50 amol was achieved for the tryptic digests of standard glycoprotein (IgG) and 1576 glycosylation sites from 713 glycopro-teins were feasibly identified from only 60 μg mouse liver proteins