Single-stranded DNA as a cleavable linker for bioorthogonal click chemistry-based proteomics

Area of Science:

• Biochemistry
• Molecular Biology
• Glycomics

Background:

• Glycoproteins play vital roles in numerous biological processes.
• Efficient methods for isolating and identifying glycoproteins are crucial for understanding cellular functions and diseases.
• Existing enrichment techniques may have limitations in selectivity or require harsh release conditions.

Purpose of the Study:

• To develop and validate a novel cleavable linker system for glycoprotein enrichment.
• To assess the efficiency and selectivity of the DNA-based linker for capturing alkyne-tagged glycoproteins.
• To identify sialylated glycoproteins from mammalian cell lysates using the developed method.

Main Methods:

• Synthesis of a biotin-DNA-azide (biotin-DNA-N3) probe for glycoprotein capture.
• Affinity enrichment of alkyne-tagged glycoproteins from mammalian cell lysates using the probe and streptavidin agarose.
• Selective release of captured glycoproteins via DNase treatment.
• Identification of released glycoproteins using mass spectrometry.

Main Results:

• The biotin-DNA-N3 probe efficiently enriched alkyne-tagged glycoproteins.
• DNase treatment provided highly efficient and selective release of captured proteins under mild conditions.
• A total of 36 sialylated glycoproteins were identified from HL60 cell lysates.
• Identified glycoproteins are involved in diverse biological processes, including glycan biosynthesis and cell adhesion.

Conclusions:

• A novel class of cleavable linkers based on single-stranded DNA has been successfully developed.
• This DNA-based enrichment strategy offers a mild and efficient method for glycoprotein analysis.
• The method facilitates the identification of key glycoproteins involved in cellular functions and disease states, such as leukemia.