Characterizing recombinant protein and its fragmentation by top-down mass spectrometry

Area of Science:

• Analytical Chemistry
• Biochemistry
• Proteomics

Background:

• Recombinant proteins are crucial therapeutics, requiring robust characterization.
• Bottom-up mass spectrometry methods face challenges with repetitive sequences and intact protein analysis.
• Top-down mass spectrometry (TDMS) offers an alternative for comprehensive protein analysis.

Purpose of the Study:

• To establish and validate TDMS workflows for evaluating structural integrity and fragmentation profiles of recombinant proteins.
• To compare targeted MS²-based and data-dependent acquisition (DDA)-based TDMS strategies.
• To provide insights into identifying proteins below 50 kDa using TDMS.

Main Methods:

• Development of targeted MS²-based and DDA-based TDMS workflows.
• Analysis of three representative recombinant proteins: RC1, RC2, and interferon α2b.
• Utilized Higher-energy collisional dissociation (HCD) fragmentation and disulfide reduction pretreatment.

Main Results:

• TDMS enabled direct sequencing of proteins with repetitive sequences, overcoming bottom-up limitations.
• DDA-based TDMS achieved high-throughput identification for proteins <12 kDa (50% fragment coverage for RC1).
• Target-MS²-based TDMS improved sequence coverage for proteins >12 kDa (up to 49.5% for RC2).
• Disulfide bond pretreatment was essential for proteins with disulfide bonds to enhance cleavage coverage.

Conclusions:

• TDMS provides complementary data to bottom-up methods for comprehensive recombinant protein characterization.
• The developed TDMS strategies are effective for proteins below 50 kDa, including those with disulfide bonds.
• This work enhances the analytical toolkit for quality control and development of therapeutic proteins.