29-Plex tandem mass tag mass spectrometry enabling accurate quantification by interference correction

Area of Science:

• Proteomics
• Mass Spectrometry
• Chemical Biology

Background:

• Tandem Mass Tag (TMT) mass spectrometry is a key isobaric chemical labeling technique for proteome profiling.
• Existing TMT multiplexing strategies (e.g., 11-plex, 18-plex) face limitations in throughput and quantitative accuracy due to ion interference.

Purpose of the Study:

• To develop and validate a novel 29-plex TMT method by integrating 11-plex and 18-plex labeling strategies.
• To address and correct for ratio compression caused by co-isolated TMT-labeled ions (noise) in high-plex TMT experiments.
• To enhance the throughput and quantitative accuracy of proteomic profiling using TMT mass spectrometry.

Main Methods:

• A 29-plex TMT method was created by combining 11-plex and 18-plex labeling kits.
• Experimental validation involved analyzing pooled Escherichia coli peptides against a human peptide background at varying ratios (1×, 3×, 10×).
• Noise detection utilized unique reporter ion features in MS/MS spectra arising from the mixture of TMT sets, followed by noise level estimation and reporter ion intensity correction.

Main Results:

• The combined 29-plex TMT method, without correction, exhibited ratio compression (e.g., 1.0:1.7:4.2) compared to expected ratios (1:3:10) due to co-isolated ion noise.
• Distinct spectral features in TMT11 and TMT18 datasets allowed for the identification and estimation of noise contributions.
• Post-correction of reporter ion intensities largely restored the anticipated quantitative ratios, validated with a 1:5 ratio sample.

Conclusions:

• The developed 29-plex TMT strategy significantly expands multiplexing capacity in proteomic analyses.
• The noise correction method effectively mitigates ratio compression, leading to improved quantitative accuracy in high-plex TMT experiments.
• This enhanced TMT approach offers a robust solution for high-throughput, accurate proteome profiling.