A Triple Knockout (TKO) Proteomics Standard for Diagnosing Ion Interference in Isobaric Labeling Experiments

Isobaric labeling is a powerful strategy for quantitative mass spectrometry-based proteomic investigations. A complication of such analyses has been the co-isolation of multiple analytes of similar mass-to-charge resulting in the distortion of relative protein abundance measurements across samples. When properly implemented, triple-stage mass spectrometry and synchronous precursor selection (SPS-MS3) can reduce the occurrence of this phenomena, referred to as ion interference. However, no diagnostic tool is available currently to rapidly and accurately assess ion interference. To address this need, we developed a multiplexed TMT-based standard, termed the triple knockout (TKO). This standard is comprised of three yeast proteomes in triplicate, each from a strain deficient in a highly abundant protein (Met6, Pfk2, or Ura2). The relative abundance patterns of these proteins, which can be inferred from dozens of peptide measurements, are representative of ion interference in peptide quantification. We expect no signal in channels where the protein is knocked out, permitting maximum sensitivity for measurements of ion interference against a null background. Here, we emphasize the need to investigate further ion interference-generated ratio distortion and promote the TKO standard as a tool to investigate such issues.