An integrated top-down and bottom-up strategy for broadly characterizing protein isoforms and modifications

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Traditional bottom-up proteomics struggles with characterizing multiple protein isoforms and genetic variants.
• Top-down proteomics offers intact protein analysis but often suffers from low throughput.
• A need exists for a comprehensive, high-throughput method for intact protein profiling.

Purpose of the Study:

• To develop and demonstrate an integrated top-down and bottom-up approach for comprehensive, high-throughput intact protein profiling.
• To overcome the limitations of conventional bottom-up and top-down proteomics methods.
• To identify protein isoforms, including those resulting from amino acid modifications and genetic variants.

Main Methods:

• Concurrent liquid chromatography-mass spectrometry (LC-MS) analysis with fraction collection.
• High-resolution, reversed-phase (RP) LC coupled online with a 12 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer.
• Integration of intact protein masses from top-down analysis with peptide identifications from bottom-up analysis of LC fractions.
• Off-line gas-phase fragmentation of selected intact proteins.

Main Results:

• Successful profiling and tentative identification of modified proteins and genetic variants.
• Demonstration of identifying protein isoforms arising from acetylation, phosphorylation, and single nucleotide polymorphisms (SNPs).
• Validation of the integrated approach in a proof-of-principle study.

Conclusions:

• The integrated top-down and bottom-up strategy provides a comprehensive and high-throughput solution for intact protein profiling.
• This approach enhances the characterization of protein modifications and genetic variations.
• The method offers significant advantages over traditional proteomics techniques for complex biological samples.