Indirect ultrasonication for protein quantification and peptide mass mapping through mass spectrometry-based...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Accurate protein quantification and peptide mass mapping are crucial in proteomics.
• Existing methods can be time-consuming and require frequent recalibration.
• Isotopic labeling offers a pathway to improved accuracy and efficiency.

Purpose of the Study:

• To develop a fast and efficient protocol for protein quantification and peptide mass mapping.
• To investigate the impact of trypsin purity and pH on (18)O labeling efficiency.
• To establish the utility of a single calibration curve for protein quantification over time.

Main Methods:

• Utilized oxygen-18 (¹⁸O) isotopic labeling via a decoupling procedure.
• Optimized the pH for efficient double (18)O labeling.
• Established and validated a calibration curve for matrix-assisted laser desorption/ionization (MALDI) protein quantification.
• Applied the method to quantify Bovine Serum Albumin (BSA) and generate peptide mass maps from fish plasma (Cyprinus carpio).

Main Results:

• Demonstrated that trypsin purity and source do not affect labeling efficiency.
• Identified pH as a critical factor for significant (18)O double labeling.
• Showed that a single calibration curve is accurate for MALDI protein quantification over several days.
• Successfully performed protein quantification and peptide mass mapping using the developed protocol.

Conclusions:

• The developed (18)O labeling protocol offers a fast and simplified approach for protein quantification and peptide mass mapping.
• The method demonstrates robustness and reproducibility, reducing the need for daily recalibration.
• This technique provides a valuable tool for routine proteomics applications.