Improved isobaric tandem mass tag quantification by ion mobility mass spectrometry

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Isobaric tandem mass tags (TMTs) offer high multiplexing for quantitative proteomics but can produce chimeric MS/MS spectra.
• Chimeric spectra lead to underestimation of peptide fold-change expression, impacting quantitative accuracy.
• Ion mobility (IM) coupled with MS/MS shows potential to resolve these spectral interferences.

Purpose of the Study:

• To evaluate the effectiveness of ion mobility (IM) separation in mitigating isobaric interference in TMT-labeled quantitative proteomics.
• To assess the impact of IM separation on the accuracy of reporter ion quantification.

Main Methods:

• Analysis of complex protein mixtures labeled with DiLeu TMTs using LC/IM-MS/MS.
• Comparison of reporter ion accuracy with and without IM separation for interfering peptide pairs.

Main Results:

• IM separation effectively reduced isobaric interference from both differentially charged and same-charged interfering ion pairs.
• Quantitative accuracy of reporters was significantly improved post-IM separation.
• Only one of eight example precursors showed residual compression below the significance threshold after IM separation.

Conclusions:

• Ion mobility separation prior to MS/MS fragmentation is a viable strategy to mitigate quantitative inaccuracies caused by isobaric interference.
• This proof-of-principle study highlights IM-MS/MS as a valuable tool for accurate TMT-based proteomics.
• Future work should focus on automated software correction and higher-resolution IM instrumentation.