Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

Area of Science:

• Proteomics
• Biomarker Discovery
• Quantitative Biology

Background:

• Increasing demand for analyzing multiple biological samples for disease understanding and biomarker discovery.
• Quantitative proteomics strategies enable simultaneous measurement of multiple samples, reducing costs and time.
• Traditional isotopic labeling or isobaric tagging approaches have limitations in sample multiplexing.

Purpose of the Study:

• To introduce and validate a novel technique, combined precursor isotopic labeling and isobaric tagging (cPILOT), for enhanced sample multiplexing in quantitative proteomics.
• To demonstrate the application of global cPILOT for analyzing protein abundances across diverse biological conditions.
• To showcase a 12-plex analysis of mouse tissues using cPILOT.

Main Methods:

• cPILOT combines low pH selective N-terminal dimethylation with high pH lysine residue labeling using isobaric reagents.
• The degree of multiplexing depends on the number of precursor labels and isobaric tagging reagents used.
• A 12-plex analysis was performed on mouse brain, heart, and liver tissues using light/heavy dimethylation and six-plex isobaric reagents.

Main Results:

• cPILOT successfully enabled a 12-plex quantitative proteomics analysis of mouse tissues in a single experiment.
• The approach allows for relative protein abundance measurements across multiple sample conditions, including disease models.
• Demonstrated application in comparing Alzheimer's disease mouse models with wild-type controls.

Conclusions:

• Global cPILOT significantly enhances sample multiplexing capabilities in quantitative proteomics.
• This method reduces experimental time and cost while minimizing experimental bias and error.
• cPILOT is applicable to various biological samples and can be adapted for multiplexing over 20 samples for broader biological studies.