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Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies
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Method of Monitoring the Number of Amide Bonds in Peptides Using Near-Infrared Spectroscopy
Mika Ishigaki1,2, Atsushi Ito3, Risa Hara3
1Institute of Agricultural and Life Sciences, Academic Assembly, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan.
Analytical Chemistry
|December 28, 2020
Summary
Near-infrared (NIR) spectroscopy can monitor peptide synthesis by tracking amide bonds. This method accurately quantifies amide bonds, offering a valuable tool for process analytical technology in peptide production.
Area of Science:
- Analytical Chemistry
- Spectroscopy
- Biochemistry
Background:
- Peptide synthesis involves forming amide bonds through dehydration.
- Monitoring amide bond formation is crucial for process control.
- Polyglycine serves as a model for studying polyamino acid chain elongation.
Purpose of the Study:
- To develop a near-infrared (NIR) spectroscopy method for monitoring amide bond formation during peptide synthesis.
- To identify key spectral bands correlating with peptide chain length.
- To establish a quantitative model for predicting amide bond numbers.
Main Methods:
- Near-infrared (NIR) spectroscopy was employed to analyze polyglycine.
- Key absorption bands in the 5000-4500 cm⁻¹ region were identified.
- Partial least squares regression was used to build a calibration model.
Main Results:
- Specific combination bands of amide A and amide II/III modes showed increased intensity with peptide chain length.
- Second derivative intensities of these bands correlated directly with the number of amide bonds.
- A robust calibration model (R² ≥ 0.99) was developed for quantifying amide bonds.
- The method proved effective regardless of pH, solvent, or protecting groups.
Conclusions:
- NIR spectroscopy is a viable process analytical technology for monitoring peptide synthesis in microflow reactors.
- The identified spectral features provide fundamental data for analyzing amide bonds in various biomolecules and polymers.
- This technique offers a non-invasive and efficient approach for real-time synthesis monitoring.
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