Differentially isotope-coded N-terminal protein sulphonation: combining protein identification and quantification

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Existing proteomic labeling techniques primarily focus on enhancing protein quantification or peptide sequencing.
• There is a continuous need for innovative methods that offer simultaneous identification and quantification capabilities for complex biological samples.

Purpose of the Study:

• To develop and validate a novel stable-isotope labeling strategy for simultaneous protein identification and quantification.
• To utilize 4-sulphophenyl isothiocyanate (SPITC) for specific N-terminal derivatization and differential isotope coding.
• To assess the applicability of this method across different mass spectrometry platforms.

Main Methods:

• N-terminal derivatization of proteins using unlabeled and 13C-labeled 4-sulphophenyl isothiocyanate (SPITC).
• Differential isotope coding with a 6 Da spacing between peptide pairs.
• Optimization of SPITC reaction conditions using three model proteins.
• Analysis of derivatized peptides using negative-ion MALDI-MS and LC-ESI-MS/MS.

Main Results:

• Successful simultaneous identification and quantification of proteins in complex mixtures.
• Enhanced detection of N-terminally derivatized peptides compared to native peptides, particularly in negative-ion MALDI-MS.
• Simplified fragmentation patterns of derivatized peptides, facilitating protein identification.
• Effective relative quantification using MALDI-TOF/TOF and LC-ESI-MS/MS due to convenient peptide pair spacing and lack of isotopic overlap.

Conclusions:

• The developed SPITC-based N-terminal tagging method offers a novel approach for quantitative proteomics.
• This technique facilitates both protein identification and quantification, improving upon existing labeling strategies.
• The method's compatibility with multiple MS platforms enhances its versatility for proteomic studies.