Measuring and Managing Ratio Compression for Accurate
iTRAQ/TMT Quantification
- Publication date
- Publisher
Abstract
Isobaric
mass tagging (e.g., TMT and iTRAQ) is a precise and sensitive
multiplexed peptide/protein quantification technique in mass spectrometry.
However, accurate quantification of complex proteomic samples is impaired
by cofragmentation of peptides, leading to systematic underestimation
of quantitative ratios. Label-free quantification strategies do not
suffer from such an accuracy bias but cannot be multiplexed and are
less precise. Here, we compared protein quantification results obtained
with these methods for a chemoproteomic competition binding experiment
and evaluated the utility of measures of spectrum purity in survey
spectra for estimating the impact of cofragmentation on measured TMT-ratios.
While applying stringent interference filters enables substantially
more accurate TMT quantification, this came at the expense of 30%–60%
fewer proteins quantified. We devised an algorithm that corrects experimental
TMT ratios on the basis of determined peptide interference levels.
The quantification accuracy achieved with this correction was comparable
to that obtained with stringent spectrum filters but limited the loss
in coverage to <10%. The generic applicability of the fold change
correction algorithm was further demonstrated by spiking of chemoproteomics
samples into excess amounts of <i>E. coli</i> tryptic digests