Multiplex proteomics using isobaric labeling tags has emerged as a powerful tool for the simultaneous relative quantification of peptides and proteins across multiple experimental conditions. However, the quantitative accuracy of the approach is largely compromised by a phenomenon termed ion interference, causing fold changes to appear compressed. The degree of compression is generally unclear, and the contributing factors are poorly understood. In this study, we thoroughly characterize ion

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... rference at the MS2 level by using a defined two-proteome experimental system. We address the poor agreement between the apparent precursor purity in the isolation window and the actual level of interference in MS2-scans, a discrepancy that is only effectively resolved by considering potential co-fragmenting peptide ions hidden within the noise. We further propose a comprehensive modeling strategy for obtaining accurate, feature-wise assessments of interference. Finally, we present a simple algorithm to calculate interference-corrected reporter ion intensities of peptides and proteins, thereby successfully removing ratio compression.