Top-Down Analysis of Supercharged Proteins Using Collision-, Electron-, and Photon-Based Activation Methods.

Area of Science:

• Proteomics
• Mass Spectrometry
• Biochemistry

Background:

• Supercharging increases protein charge states, potentially altering fragmentation behavior in mass spectrometry.
• Understanding these changes is crucial for optimizing protein analysis and sequence coverage.

Purpose of the Study:

• To investigate the impact of supercharging on protein fragmentation patterns using various activation methods.
• To evaluate changes in sequence coverage and preferential cleavage sites under different activation conditions.

Main Methods:

• Six model proteins were analyzed using five activation methods: Higher-energy collisional dissociation (HCD), Electron Transfer Dissociation (ETD), Electron Transfer Higher-energy Collisional Dissociation (EThcD), 213 nm Ultraviolet Photodissociation (UVPD), and 193 nm UVPD.
• Proteins were analyzed under both non-supercharged and supercharged conditions.
• Fragmentation patterns, sequence coverage, and cleavage site preferences were evaluated.

Main Results:

• Supercharging led to significant decreases in sequence coverage with HCD, but modest gains with ETD.
• EThcD, 213 nm UVPD, and 193 nm UVPD showed minimal changes in sequence coverage, generally yielding the highest coverages.
• Preferential backbone cleavage sites were enhanced across all methods, especially with HCD, 213 nm UVPD, and 193 nm UVPD.
• Supercharging consistently introduced new backbone cleavage sites for ETD, EThcD, 213 nm UVPD, and 193 nm UVPD.

Conclusions:

• The effect of supercharging on protein fragmentation is highly dependent on the activation method used.
• UVPD and EThcD are robust methods for maintaining high sequence coverage even with supercharged proteins.
• Supercharging can enhance the identification of specific cleavage sites, aiding in detailed proteomic analysis.