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
Sample Processing Protocol
Enrichment of glycopeptides by the homogeneous reaction system Step 1: Preparation of 1% TFA (ACN), the aqueous solution of Au NCs and tryptic protein sample. Step 2: 40 µL Au NCs and 3 µL protein sample were mixed together and shocked at 1050 rpm for 2 min. Step 3: 356 µL 1% TFA (ACN) were added dropwise in about 30 s and the mixture were furtherly shocked for 30 s. Then the Au NCs were collected by centrifugation. Step 4 for washing: The precipitated Au NCs were re-dissolved into 40 µL pure H2O and the solution were shocked at 1050 rpm for 2 min. Step 5: Repeat the Step 3. Step 6: Repeat the Step 4-5 for another 1-2 times. Step 7 for elution: The Au NCs were dispersed in 20 µL 30%ACN/0.1% TFA solution and shocked for 5 min. After centrifugation, the supernatant was collected and lyophilized for MS analyses.
Data Processing Protocol
The LC-MS/MS raw data files were analyzed with Maxquant software (version 220.127.116.11) against a UniProt.mouse.fasta database (downloaded on April 9, 2014) with the default settings. Only glycopeptides with the N-!P-S/T or N-X-C conserved sequences were considered as highly reliable results.
Li J, Liu J, Liu Z, Tan Y, Liu X, Wang F. Detecting Proteins Glycosylation by a Homogeneous Reaction System with Zwitterionic Gold Nanoclusters. Anal Chem. 2017 Mar 27 PubMed: 28345880