TKO6: A Peptide Standard to Assess Interference for Unit-Resolved Isobaric Labeling Platforms

Protein abundance profiling using isobaric labeling is a well-established quantitative mass spectrometry technique. However, ratio distortion resulting from co-isolated and co-fragmented ions - commonly referred to as interference - remains a caveat of this technique. Tribrid mass spectrometers, such as the Orbitrap Fusion and the Orbitrap Fusion Lumos with a triple mass analyzer configuration, facilitate methods (namely SPS-MS3) that can help alleviate interference. Yet, few standards are available to measure interference. Here we introduce the TKO6 standard that assesses ion interference and is designed specifically for data acquired at low (unit) mass resolution. We use TKO6 to compare the degree of interference in MS2 versus MS3-based quantitation methods, data acquisition methods of different lengths, and ion trap-based TMT reporter ion analysis (IT-MS3). We show that the TKO6 standard is a valuable tool for assessing data quality and is particularly useful for benchmarking ion trap-based SPS-MS3 analyses.