Global quantitative proteomic profiling through 18O-labeling in combination with MS/MS spectra analysis.

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Stable-isotope labeling enables relative protein quantitation in mass spectrometry.
• Current methods often use MS level quantitation, which can be limited.
• 18O-based labeling is a common technique for comparative proteomics.

Purpose of the Study:

• To demonstrate the accuracy and sensitivity of MS/MS level quantitation for 18O-based labeling.
• To introduce software tools (ySelect, yRatios) for streamlined data analysis.
• To validate a robust workflow for comparative proteomic quantitation.

Main Methods:

• Utilized trypsin-catalyzed 18O-based peptide labeling.
• Performed quantitation at the product ion (MS/MS) level.
• Developed and applied specialized software (ySelect, yRatios) for data analysis and ratio determination.
• Validated the approach using simple and complex protein mixtures with LC-MS/MS.

Main Results:

• MS/MS level quantitation demonstrated high accuracy, sensitivity, and signal-to-noise.
• Software tools enabled accurate protein ratio determination with correction for incomplete labeling.
• Ratio distributions approached expected means, allowing for statistically significant fold-change determination.
• Stringent criteria identified and minimized spurious ratios.

Conclusions:

• MS/MS quantitation combined with 18O-labeling offers a reliable method for comparative proteomics.
• The developed software toolkit facilitates routine and accurate analysis of proteomic data.
• This approach optimizes conditions for robust protein inference and identification of significant abundance changes.