Project PXD005635

PRIDE Assigned Tags:
Biomedical Dataset

Summary

Title

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

Description

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 1.3.0.5) 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.

Contact

Jinan Li, Dalian Institute of Chemical Physics
Fangjun Wang, Dalian Institute of Chemical Physics ( lab head )

Submission Date

03/01/2017

Publication Date

29/03/2017

Tissue

liver

Instrument

Q Exactive

Software

Not available

Modification

deamidated residue

Quantification

Not available

Experiment Type

Shotgun proteomics

Publication

    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