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Easy, Robust, and Repeatable Online Acid Cleavage of Proteins in Mobile Phase for Fast Quantitative LC-MS Bottom-Up
Denis K Naplekov1, Siddharth Jadeja1, Alena Myslivcová Fučíková2
1Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203/8, 500 05 Hradec Králové, Czech Republic.
Analytical Chemistry
|August 11, 2023
Summary
This study introduces a rapid online method for protein cleavage into peptides, significantly reducing preparation time for proteomics. The technique allows for fast, accurate protein analysis and detection, streamlining workflows.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Protein sample preparation, including protein cleavage into peptides, is crucial for bottom-up proteomics.
- Current methods are often time-consuming and labor-intensive, posing bottlenecks in proteomic workflows.
Purpose of the Study:
- To develop a fast, online method for post-injection acid cleavage of proteins directly in the liquid chromatography-mass spectrometry (LC-MS) mobile phase.
- To enable rapid peptide generation and separation for efficient proteomic analysis.
Main Methods:
- Proteins were cleaved using 0.1% formic acid in a heated capillary (195 °C) positioned before the analytical column.
- The online cleavage was optimized for speed (35 s) and efficiency, integrated into standard LC-MS workflows.
Main Results:
- The method generated peptides covering nearly the entire sequence of trastuzumab, demonstrating high cleavage efficiency.
- Qualitative results showed excellent long-term stability, and the method proved suitable for accurate, repeatable quantification.
- Demonstrated practical utility by enabling unambiguous ricin detection from a 10 ng injection within 7-8 minutes.
Conclusions:
- The developed online acid cleavage method significantly accelerates protein sample preparation for proteomics.
- The technique is robust, repeatable, and suitable for both qualitative and quantitative proteomic analyses.
- Its simplicity and lack of specialized requirements facilitate seamless adoption in high-throughput laboratories.
