Establishing a Top-Down Proteomics Platform on a Time-of-Flight Instrument with Electron-Activated Dissociation

Area of Science:

• Mass Spectrometry
• Proteomics
• Biochemistry

Background:

• Top-down proteomics analyzes intact proteins and post-translational modifications (PTMs), offering advantages over bottom-up approaches.
• Historically, top-down proteomics faced challenges due to large protein sizes and complex modification combinations, necessitating technological advancements.
• Recent improvements in mass spectrometry instrumentation and fragmentation techniques are driving renewed interest and application of top-down proteomics.

Purpose of the Study:

• To systematically evaluate the Sciex ZenoTOF 7600 system for top-down proteomics applications.
• To validate the platform's performance using established standards in the field.
• To explore the system's capabilities in electron-activated dissociation (EAD) and PTM site localization.

Main Methods:

• Evaluation of the Sciex ZenoTOF 7600 system using standard proteins for quality control (QC).
• Application of zeno trapping for enhanced ion manipulation and detection.
• Utilized optimized electron-activated dissociation (EAD) fragmentation for PTM site localization.
• Analysis of histone PTMs and glycosylated proteins across a broad mass range.

Main Results:

• The Sciex ZenoTOF 7600 system demonstrated robust performance for standard proteins, confirmed by zeno trapping.
• High sequence coverage was achieved for histone PTM analysis, enabling precise PTM site localization.
• The platform successfully analyzed proteins across a wide mass range, including glycosylated proteins.
• Optimized EAD fragmentation proved effective for PTM site localization.

Conclusions:

• The Sciex ZenoTOF 7600 system is a validated and capable platform for top-down proteomics.
• The instrument facilitates high-confidence identification and quantification of intact proteins and their modifications.
• This study provides a reference point for future top-down proteomics research utilizing the ZenoTOF 7600 system.