Biofunctionalized dissolvable hydrogel microbeads enable efficient characterization of native protein complexes

Area of Science:

• Biochemistry
• Proteomics
• Molecular Biology

Background:

• Protein complex characterization is crucial for understanding biological processes.
• Analyzing low-input samples and non-covalent complexes remains challenging.
• Existing methods struggle with efficiency and accuracy for complex biochemical compositions.

Purpose of the Study:

• To introduce SNAP-MS (Stationary-phase-dissolvable Native Affinity Purification and Mass Spectrometric characterization).
• To enable highly efficient purification and characterization of protein complexes from low-input samples (pico-mole level).
• To provide a rapid and accurate method for native mass spectrometry analysis.

Main Methods:

• Developed SNAP-MS, a strategy involving stationary-phase dissolving for target recovery.
• Utilized high-affinity bait-target pairs for efficient capture.
• Integrated native mass spectrometry (MS) for structural and compositional analysis.
• Employed an algorithm for charge removal and mass correction in MS analysis.

Main Results:

• Achieved highly efficient purification and characterization from pico-mole level inputs.
• Enabled native MS analysis of intact protein complexes, revealing stoichiometry, topology, and proteoform distribution.
• Demonstrated enhanced accuracy for heterogeneously glycosylated complexes.
• Reduced sample-to-data acquisition time to as little as 2 hours.

Conclusions:

• SNAP-MS offers a versatile and efficient solution for characterizing native protein complexes.
• The method is suitable for low-input biological samples, including blood.
• SNAP-MS significantly advances the ability to study complex biological mechanisms through protein interaction analysis.