Massively parallel assessment of designed protein solution properties using mass spectrometry and peptide barcoding

Area of Science:

• Biochemistry
• Proteomics
• Molecular Biology

Background:

• Traditional library screening methods link proteins to DNA for identification but alter solution properties.
• Mass spectrometry (MS) can assess protein solution properties but is limited to small libraries (<1,000 proteins).

Purpose of the Study:

• To develop an improved MS-based method for analyzing solution properties of large protein libraries.
• To scale MS-based protein analysis to over 5,000 proteins.

Main Methods:

• Co-synthesized proteins with optimized, highly multiplexable, and minimally perturbative barcodes.
• Utilized enhanced MS barcoding to assay solution behavior (solubility, oligomeric state) of large protein libraries.
• Applied the method to de novo-designed protein scaffolds, oligomers, binding proteins, and nanocages.

Main Results:

• Successfully scaled MS-based analysis to libraries exceeding 5,000 proteins.
• Identified design failure modes and successes in diverse protein libraries.
• Enabled rapid, parallel evaluation of protein solution properties.

Conclusions:

• Enhanced MS barcoding significantly expands the scale of protein solution property analysis.
• This method accelerates the characterization and optimization of protein designs.
• Provides a powerful tool for studying protein behavior in solution.