Protein Function Scanning by Mass Spectrometry

High throughput, highly controlled mutagenesis method to study protein function of full-length proteins for protein engineering and testing, drug and antibody development, protein interaction and binding studies.
Problem:
Understanding a protein's functions requires studying each mutation separately through separation-of-function mutations. Traditional methods, such as point mutagenesis, are costly, labor-intensive, and lack the resolution and throughput needed to systematically identify relevant mutations. Further limitations of currently used methods include selection biases, size constraints, and difficulty in scanning the entire sequence of large proteins.
Solution:
The inventors developed a high-throughput mutagenesis method called ProMScan (Protein function Scanning by Mass Spectrometry) which pairs mutants along the length of a protein of any size to peptide barcodes to be scanned and functionally assessed, recovering the affected mutants by mass spectrometry. This method mitigates the representation bias through capture-based balancing of the mutant libraries and using dual barcoding system. The method can be used to study any length protein sequence.
Applications:

• Protein engineering
• Functional protein activity testing
• Drug and antibody development
• Protein-protein interaction studies
• Protein binding studies (DNA binding, RNA binding, antigen/protein/peptide binding)

Advantages:

• High throughput
• Highly controlled
• Functional assessment of any length protein in a single sample
• Can be used with bacterial, mammalian, or lentiviral expression vectors
• Enabled for both in vitro and in vivo phenotype validation

Stage of Development:

• Proof of Concept

Overview of the ProMScan method. 1) Barcoded mutant gene libraries are constructed by high throughput overlap extension PCR. 2) Next, the barcode mutation assignments are verified by nanopore sequencing. 3) The pooled variants can either be cloned into a vector and expressed as protein (3a) or transfected into cells (3b) for in vitro or in vivo applications, respectively. 4) Following phenotypic selection, 5) the peptide barcodes and its peptide intensity are read out by mass spectrometry.
Intellectual Property:

• Provisional Filed

Desired Partnerships:

• License
• Co-development

Docket: 23-10380