Systematic evaluation of label-free and super-SILAC quantification for proteome expression analysis

Area of Science:

• Proteomics
• Mass Spectrometry
• Biomarker Discovery

Background:

• Mass spectrometry (MS)-based proteomics enables comprehensive proteome profiling.
• Accurate quantification is crucial for high-coverage proteome analysis on modern instruments like quadrupole orbitrap mass spectrometers.

Purpose of the Study:

• To systematically compare label-free proteome quantification with stable isotope labeling using amino acids in cell culture (super-SILAC).
• To evaluate label-free quantification strategies for high-coverage proteome analysis on ultra-high-performance liquid chromatography (UHPLC)/MS.

Main Methods:

• UHPLC/MS experiments were performed on a Q Exactive instrument.
• Label-free quantification was compared against super-SILAC using six human cancer cell lines.
• Peptide fractionation using high pH reversed-phase chromatography was investigated.

Main Results:

• Label-free methods identified more proteins (approx. 5000) than super-SILAC (approx. 3500).
• Label-free quantification showed slightly lower precision than super-SILAC but was improved by replicate analyses and peptide fractionation.
• More significant differences in cell line comparisons were detected using label-free quantification.

Conclusions:

• Label-free proteome quantification is a valuable tool for target and biomarker discovery.
• State-of-the-art UHPLC/MS workflows benefit from robust label-free quantification strategies.
• Optimized label-free approaches enhance proteome coverage and precision.