Protein identification and quantification by two-dimensional infrared spectroscopy: implications for an all-optical...

Area of Science:

• Analytical Chemistry
• Biophysics
• Spectroscopy

Background:

• Proteomics requires sensitive and rapid methods for protein identification and quantification.
• Existing techniques may lack the speed or specificity for high-throughput applications.
• Two-dimensional infrared (2DIR) spectroscopy offers unique vibrational information about molecular structure.

Purpose of the Study:

• To evaluate electron-vibration-vibration two-dimensional coherent spectroscopy as a tool for protein differentiation and quantification.
• To identify and utilize unique 2DIR vibrational signatures of amino acid side chains.
• To assess the potential for a high-throughput proteomic platform based on 2DIR.

Main Methods:

• Utilized electron-vibration-vibration two-dimensional coherent spectroscopy (a variant of 2DIR).
• Identified 2DIR vibrational signatures specific to amino acid side chains.
• Employed a methyl vibrational feature as an internal reference for signal quantification.
• Applied the method to differentiate a set of 10 proteins based on amino acid composition.

Main Results:

• Successfully differentiated 10 proteins based on their amino acid content using 2DIR signatures.
• Quantified amino acid levels by correlating signal strengths with a methyl vibrational reference.
• Achieved protein differentiation in 4-5 minutes per sample, with potential for <1 minute.
• Demonstrated straightforward absolute quantification of protein levels.

Conclusions:

• Electron-vibration-vibration 2DIR spectroscopy is a powerful tool for protein analysis.
• The method allows for rapid differentiation and quantification of proteins based on amino acid composition.
• This technology holds significant potential for developing an all-optical, high-throughput proteomic platform.