Intact protein separation by chromatographic and/or electrophoretic techniques for top-down proteomics

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Mass spectrometry (MS) is a key proteomics tool.
• Proteomics employs bottom-up or top-down approaches.
• Top-down proteomics analyzes intact proteins using MS.

Purpose of the Study:

• To review current protein separation methods for top-down proteomics.
• To highlight chromatographic and electrophoretic techniques.
• To discuss advancements in protein separation for MS.

Main Methods:

• Top-down mass spectrometry with high-accuracy mass analyzers (e.g., LTQ-FTICR, LTQ-Orbitrap).
• Electrospray ionization (ESI) for ion generation.
• Gas-phase separation and fragmentation of protein ions.
• Automated interpretation of MS and chromatographic data.
• Monodimensional and multidimensional liquid chromatography-mass spectrometry (LC-MS).

Main Results:

• Top-down proteomics yields intact protein molecular weight and fragmentation patterns.
• High-accuracy mass analyzers are crucial for resolving multi-charged isotopic clusters.
• Effective front-end separation technologies are vital for top-down proteomics.
• Review covers state-of-the-art chromatographic and electrophoretic protein separation methods.

Conclusions:

• Advanced separation techniques are critical for successful top-down proteomics.
• LC-MS top-down approaches benefit from monodimensional and multidimensional strategies.
• Ongoing progress in protein chromatography offers alternative separation solutions.