Determining synthetic failures in combinatorial libraries by hybrid gas-phase separation methods.

Area of Science:

• Analytical Chemistry
• Mass Spectrometry
• Organic Chemistry

Background:

• Combinatorial chemistry enables the synthesis of large compound libraries.
• Analyzing complex mixtures of peptides presents significant analytical challenges.
• Ion mobility separation offers a new dimension for analyzing isomeric compounds.

Purpose of the Study:

• To develop and validate a high-resolution ion mobility/time-of-flight mass spectrometry method for analyzing a combinatorial tripeptide library.
• To demonstrate the capability of this method in identifying synthetic failures within complex peptide mixtures.
• To assess the utility of the method for improving the reliability of binding affinity studies.

Main Methods:

• Synthesis of a 676-component combinatorial tripeptide library (D-Glu-Xxx-Xxx-CONH2) using a mix-and-split protocol.
• Analysis of the peptide library using electrospray ionization coupled with high-resolution ion mobility/time-of-flight mass spectrometry.
• Separation of peptide ions based on gas-phase mobility prior to mass-to-charge ratio analysis.

Main Results:

• The high-resolution ion mobility/time-of-flight mass spectrometry method successfully resolved various sequence, structural, and stereo isomers within the tripeptide library.
• Analysis of isomer trends at different m/z ratios allowed for the identification of specific failed synthetic steps, even in complex mixtures.
• The method provided information on the relative abundances of different isomers, crucial for accurate binding studies.

Conclusions:

• High-resolution ion mobility/time-of-flight mass spectrometry is a powerful tool for analyzing complex peptide libraries.
• This technique enables the identification of synthetic issues in combinatorial synthesis, enhancing quality control.
• The ability to quantify isomer abundances significantly improves the reliability of direct binding affinity studies from mixtures.