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

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Isobaric labeling is crucial for quantitative mass spectrometry-based proteomics.
• Ion interference, caused by co-isolated analytes, distorts protein abundance measurements.
• Synchronous precursor selection and triple-stage mass spectrometry (SPS-MS3) can mitigate ion interference.

Purpose of the Study:

• To develop a diagnostic tool for assessing ion interference in quantitative proteomics.
• To introduce the triple knockout (TKO) standard for accurate ion interference evaluation.
• To address the need for reliable methods to investigate ion interference-generated ratio distortion.

Main Methods:

• Developed a multiplexed tandem mass tag (TMT)-based standard, the triple knockout (TKO).
• The TKO standard utilizes three yeast proteomes, each deficient in a highly abundant protein (Met6, Pfk2, or Ura2).
• Leveraged peptide quantification patterns from TKO strains to demonstrate ion interference against a null background.

Main Results:

• The TKO standard enables sensitive detection of ion interference by measuring deviations from expected null signals.
• Demonstrated the utility of the TKO standard in identifying and quantifying ion interference.
• Provided a method to assess the effectiveness of SPS-MS3 in reducing ion interference.

Conclusions:

• The TKO standard is a valuable tool for investigating ion interference in quantitative proteomics.
• Accurate assessment of ion interference is critical for reliable protein abundance measurements.
• Further research into ion interference is necessary to improve proteomic data quality.