Assessing the Performance of Mass Spectrometry Search Strategies in Identifying Translational Errors Using PDX...

Area of Science:

• Proteomics and Bioinformatics
• Molecular Biology
• Genetics

Background:

• Translational errors (TEs) create proteins with incorrect amino acids (AAs), distinct from DNA mutations or RNA editing.
• Detecting TEs requires protein-level analysis, with mass spectrometry (MS) proteomics showing theoretical potential but uncertain feasibility.
• Current MS data analysis methods need validation for reliable TE identification.

Purpose of the Study:

• To establish a benchmark for identifying translational errors using mass spectrometry (MS) proteomics.
• To evaluate the feasibility and performance of current MS data analysis approaches for TE detection.
• To provide guidance for optimizing MS search strategies for discovering TEs.

Main Methods:

• Utilized patient-derived xenograft (PDX) proteomics data containing human and mouse peptides with cross-species AA variations.
• Employed high-confidence mouse peptides as surrogates for 'TE-containing' peptides to benchmark open search approaches.
• Analyzed the impact of intersecting different search strategies and evaluated performance metrics for specific AA substitutions.

Main Results:

• Open search approaches demonstrated over 65% sensitivity and 70% precision for TE identification in high-quality samples.
• Combining different search strategies improved precision but reduced sensitivity.
• Performance varied significantly across different amino acid substitutions, necessitating cautious interpretation.
• Closed searches showed poor precision, with post-translational modification (PTM) mislocalization being a major limitation.

Conclusions:

• This study provides the first benchmark for MS-based translational error discovery.
• Current open search strategies show promise for TE identification, especially when data quality is high.
• Optimized MS search strategies are crucial for accurate and reliable detection of translational errors, with specific AA substitutions requiring careful consideration.