Evaluation and Improvement of Quantification Accuracy in Isobaric Mass Tag-Based Protein Quantification Experiments

Area of Science:

• Proteomics
• Mass Spectrometry
• Quantitative Biology

Background:

• Isobaric mass tag-based protein quantification, like Tandem Mass Tags (TMT), enables high-throughput proteomics.
• Reporter ion quantification in TMT can suffer from accuracy issues due to coeluting interferences.
• Existing methods require improvements for reliable quantification in complex samples.

Purpose of the Study:

• To investigate quantification accuracy limitations in TMT-based proteomics.
• To propose and evaluate a novel ratio adjustment method using a calibration mixture.
• To compare TMT quantification with label-free quantification in complex biological samples.

Main Methods:

• Developed a ratio adjustment approach by spiking a 6-protein calibration mixture into samples.
• Evaluated the method on large-scale TMT 10-plex datasets from human cell lines and E. coli.
• Analyzed a complex 2-proteome artificial mixture and compared TMT with label-free quantification.

Main Results:

• The proposed calibration mixture method effectively addresses quantification accuracy issues in TMT.
• Differential protein abundance analysis showed higher statistical significance with TMT quantification.
• Demonstrated the precision of TMT and label-free quantification in complex proteomic samples.

Conclusions:

• Spiking a calibration mixture is an effective and easy-to-implement solution for improving TMT quantification accuracy.
• TMT-based quantification offers enhanced statistical power for identifying differentially abundant proteins.
• The study validates TMT as a robust technique for complex proteome analysis.