Identification of proteins bound to a thioaptamer probe on a proteomics array

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Identifying proteins interacting with specific nucleic acid sequences is crucial for understanding cellular processes.
• Existing methods for protein-nucleic acid interaction analysis can be time-consuming and require highly purified samples.

Purpose of the Study:

• To develop a rapid and efficient method for screening and identifying unknown proteins bound to nucleic acid probes on ProteinChip arrays.
• To demonstrate the utility of this method using a specific thioaptamer and mouse cell extracts.

Main Methods:

• Direct coupling of a thioaptamer (XBY-S2) to pre-activated ProteinChip array surfaces.
• Pre-fractionation of crude nuclear extracts using ion exchange chromatography.
• On-chip digestion of captured proteins followed by microsequencing via tandem mass spectrometry (MS/MS).
• Verification using a thioaptamer/antibody sandwich assay.

Main Results:

• Successfully screened and identified specific binding proteins from LPS-stimulated mouse 70Z/3 pre-B cell nuclear extracts.
• Identified five mouse heterogeneous nuclear ribonucleoproteins (hnRNPs): A1, A2/B1, A3, A/B, and D0.
• Achieved sufficient purity of "on-chip" captured proteins for on-chip digestion and identification.

Conclusions:

• The developed method provides a rapid and sensitive approach for identifying protein-nucleic acid interactions directly from crude biological samples.
• The thioaptamer/antibody sandwich assay offers a highly sensitive and selective verification method for on-chip protein identification.