A label-free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen

Area of Science:

• Proteomics
• Quantitative Biology
• Biomarker Discovery

Background:

• Accurate quantification of proteins and post-translational modifications (PTMs) is crucial for understanding cellular signaling and identifying disease biomarkers.
• Stable isotope labeling is a common quantitative proteomics technique but is limited by a dynamic range of approximately 20:1.

Purpose of the Study:

• To develop and validate a label-free proteomics method for relative protein quantification with an expanded dynamic range.
• To assess the utility of this method for identifying proteins interacting with specific targets, such as phosphotyrosine residues.

Main Methods:

• Utilized a label-free quantitative proteomics approach based on the total ion current (TIC) from peptide MS/MS spectra.
• Collected data using standard data-dependent acquisition runs on commercial mass spectrometers.
• Applied the method to screen for proteins binding to phosphotyrosine residues.

Main Results:

• The label-free TIC-based method achieved a dynamic range of up to approximately 60:1, significantly exceeding that of isotope labeling.
• Demonstrated the method's effectiveness in identifying proteins of interest in a specific biological context.

Conclusions:

• Label-free quantification using TIC in MS/MS spectra is a viable and powerful alternative to isotope labeling for relative protein quantification.
• This method expands the accessible dynamic range, enabling more comprehensive proteomic analyses and biomarker discovery.