A method combining SPITC and ¹⁸O labeling for simultaneous protein identification and relative quantification

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Protein identification and relative quantification are crucial in proteomic research.
• Current methods often require separate experiments for identification and quantification.
• Chemical N-terminal derivatization and isotopic labeling are established techniques.

Purpose of the Study:

• To develop a single method for simultaneous protein identification and quantification.
• To combine 4-sulphophenyl isothiocyanate derivatization with (18)O isotopic labeling.
• To validate the method's efficiency and applicability in complex biological samples.

Main Methods:

• Optimization of reaction conditions using standard peptides and model proteins.
• Integration of 4-sulphophenyl isothiocyanate derivatization for de novo sequencing.
• Application of (18)O labeling for relative protein quantification.
• Utilizing MS/MS spectra for both identification and quantification.

Main Results:

• Optimized conditions demonstrated good compatibility between derivatization and labeling.
• Stable linear quantification was achieved within a tenfold dynamic range (R² = 0.998).
• Accurate protein identification and quantification were achieved in a single MS/MS spectrum.
• Successful application to spider venoms, identifying differentially expressed proteins and their quantitative ratios.

Conclusions:

• The developed method enables simultaneous protein identification and quantification.
• It offers a simple, reliable, and efficient approach for proteomic analysis.
• The method shows significant potential for studying proteomes of species with unknown genomes.