Comparison of iTRAQ and SWATH in a clinical study with multiple time points

Area of Science:

• Proteomics
• Biomarker Discovery
• Mass Spectrometry

Background:

• Mass spectrometry advances biomarker discovery in medicine.
• Methodological constraints like limited sample size and multiple time points pose challenges.
• Comparing analytical efficiency of iTRAQ and SWATH methods is crucial for biomarker discovery.

Purpose of the Study:

• To compare the analytical efficiency of isobaric tags for relative and absolute quantitation (iTRAQ) and sequential window acquisition of all theoretical fragment ion spectra (SWATH) methods.
• To evaluate these mass spectrometry techniques under constraints of limited sample size and multiple time points.
• To determine the preferred method for biomarker discovery in challenging clinical sample scenarios.

Main Methods:

• Analysis of 140 tear samples from 28 glaucoma patients across 5 time points.
• Utilized NanoLC-MSTOF mass spectrometry for both iTRAQ and SWATH analyses.
• Assessed method repeatability using percent relative standard deviation (% RSD).

Main Results:

• SWATH demonstrated higher sensitivity, reliability, and robustness compared to iTRAQ.
• SWATH yielded complete data for 456 proteins, while iTRAQ quantified an average of 125 proteins per sample.
• SWATH exhibited better repeatability (56% of proteins < 20% RSD) than iTRAQ (43% of proteins < 20% RSD).
• Both methods showed similar accuracy and detected similar trends for known biomarkers.

Conclusions:

• Sequential window acquisition of all theoretical fragment ion spectra (SWATH) is recommended over isobaric tags for relative and absolute quantitation (iTRAQ) for biomarker discovery.
• SWATH is particularly advantageous when dealing with limited clinical sample volumes and multiple time points.
• The findings support SWATH as a preferred method for complex proteomic analyses in clinical research.