Mass spectrometry-based structural dissection of fluorescent proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Microscopy

Background:

• Fluorescent proteins (FPs) are crucial tools for live cell imaging.
• The molecular mechanisms behind FP photobleaching and the resulting photoproducts remain poorly understood.
• Standard mass spectrometry techniques are hindered by the inherent resistance of FPs to enzymatic digestion.

Purpose of the Study:

• To develop a novel method for analyzing the structure of fluorescent proteins.
• To overcome the limitations of trypsinolysis in mass spectrometry-based FP studies.
• To enable high-resolution mass spectrometric investigation of FP photobleaching and photoproducts.

Main Methods:

• A mild endoproteolysis procedure was developed.
• This method enhances peptide liberation from the compact FP barrel structure.
• The procedure generates a comprehensive peptide mass fingerprint.

Main Results:

• The new method significantly improves the digestibility of fluorescent proteins.
• A peptide mass fingerprint covering nearly the entire FP sequence was achieved.
• This facilitates detailed structural analysis using high-resolution mass spectrometry.

Conclusions:

• The developed mild endoproteolysis technique is effective for structural studies of FPs.
• This advancement allows for deeper insights into the photobleaching process of FPs.
• It opens new avenues for investigating FP photochemistry and developing improved FPs.