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False discovery rate estimation and heterobifunctional cross-linkers.

Lutz Fischer1, Juri Rappsilber1,2

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False discovery rate (FDR) estimation in proteomics is crucial. This study shows a single FDR formula works for both heterobifunctional and homobifunctional cross-linkers in cross-linking/mass spectrometry, simplifying data analysis.

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Biochemistry

Background:

  • False discovery rate (FDR) estimation is essential for interpreting high-throughput proteomics data.
  • Cross-linking/mass spectrometry (XL-MS) is a powerful technique for studying protein structures and interactions.
  • Current FDR estimation methods in XL-MS may not fully account for different cross-linker types.

Purpose of the Study:

  • To investigate the practical differences in FDR estimation between heterobifunctional and homobifunctional cross-linkers in XL-MS.
  • To develop and evaluate a unified FDR calculation approach for non-cleavable cross-linkers.
  • To determine if separate FDR formulas are necessary for different cross-linker chemistries.

Main Methods:

  • Theoretical evaluation of FDR calculation for heterobifunctional cross-linkers.
  • Comparative analysis of FDR estimation using distinct and unified formulas on XL-MS datasets.
  • Statistical assessment of the impact of different FDR approaches on identified cross-linked peptides.

Main Results:

  • Heterobifunctional cross-linkers do not necessitate a separate FDR calculation compared to homobifunctional cross-linkers in XL-MS.
  • A single, unified FDR formula provides minimal practical differences when applied to data from various non-cleavable cross-linkers.
  • The study validates the applicability of a generalized FDR approach across different cross-linking strategies.

Conclusions:

  • A single FDR estimation formula is sufficient and practical for analyzing data from both homo- and heterobifunctional non-cleavable cross-linkers in mass spectrometry.
  • This unification simplifies the statistical analysis pipeline for cross-linking/mass spectrometry experiments.
  • Researchers can confidently apply a consistent FDR calculation method, regardless of the specific non-cleavable cross-linker used.