A novel triplex isobaric termini labeling quantitative approach for simultaneously supplying three quantitative...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biomarker Discovery

Background:

• Isobaric tandem Mass Spectrometry (MS2) is vital for high-sensitivity, simultaneous protein biomarker screening.
• Existing MS2 methods face challenges in accurate quantification across diverse sample types and ratios.

Purpose of the Study:

• To establish and validate a novel isobaric MS2 quantification method, Triplex Quantification by Isobaric Termini Labeling (Triplex-QITL).
• To enable simultaneous comparison of multiple fragment ion types for enhanced quantitative accuracy.

Main Methods:

• Proteins digested with Lys-C, followed by N-terminal dimethylation and C-terminal mTRAQ labeling.
• Simultaneous generation of reporter ions, amino acid fragments, and N-/C-terminal fragment ion pairs in MS2 spectra.
• Comparative analysis of quantification accuracy using different fragment ion types.

Main Results:

• Triplex-QITL demonstrated higher reproducibility and accuracy compared to a1 ion quantification in SW480 cell lysates.
• N- and C-terminal ions provided more accurate quantification, especially for large ratio differences (1:10:10).
• The method successfully profiled metastatic differences in hepatocellular carcinoma (HCC) cell lines.

Conclusions:

• Triplex-QITL offers a promising advancement for quantitative proteomics.
• The method enhances accuracy and reproducibility in protein biomarker screening.
• It shows significant potential for analyzing complex biological samples and disease profiling.