Improving peptide fragmentation by N-terminal derivatization with high proton affinity

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• De novo peptide sequencing is crucial for identifying proteins.
• Mass spectrometry-based methods often benefit from enhanced fragmentation.
• N-terminal derivatization can improve peptide ion intensity and spectral interpretation.

Purpose of the Study:

• To develop an improved de novo peptide sequencing method using mass spectrometry.
• To investigate the impact of N-terminal derivatization reagents with high proton affinity on peptide fragmentation.
• To identify optimal physicochemical properties of derivatization reagents for efficient fragmentation.

Main Methods:

• Synthesis of novel N-terminal derivatization reagents with high proton affinity (guanidino or amidino moieties).
• Application of these reagents to peptides for analysis via mass spectrometry.
• Evaluation of fragmentation efficiency under low-energy collision-induced dissociation (CID) conditions.
• Comparison of spectral data to determine the effect of different derivatization reagents on b-ion intensity.

Main Results:

• Derivatization with reagents possessing high proton affinity enhanced b-ion intensity in product ion spectra.
• An optimal proton affinity was identified for efficient peptide fragmentation under low-energy CID.
• Derivatization using 4-amidinobenzoic acid yielded the most effective fragmentation among the tested reagents.
• The developed method facilitates easier interpretation of mass spectra for de novo sequencing.

Conclusions:

• Novel N-terminal derivatization reagents with high proton affinity significantly improve de novo peptide sequencing.
• The study identified 4-amidinobenzoic acid as a highly effective reagent for enhancing peptide fragmentation.
• This approach offers a promising new strategy for de novo peptide sequencing under low-energy CID conditions.