Proteome-scale recombinant standards and a robust high-speed search engine to advance cross-linking MS-based...

Area of Science:

• Biochemistry
• Proteomics
• Mass Spectrometry

Background:

• Advancing data analysis for proteome-wide cross-linking mass spectrometry (XL-MS) necessitates reliable standards.
• Existing methods lack ground-truth datasets that accurately reflect biological complexity.

Purpose of the Study:

• To develop well-controlled XL-MS standards for advancing data analysis tools.
• To introduce Scout, a novel search engine for XL-MS data, particularly for MS-cleavable cross-linkers.

Main Methods:

• Systematic mixing of hundreds of recombinant proteins to create XL-MS standards.
• Development of the Scout search engine using a dedicated standard dataset.
• Benchmarking Scout and existing XL-MS software against independent standard and published datasets.

Main Results:

• Scout demonstrated a strong performance profile, balancing speed, sensitivity, and false discovery rate control.
• The developed recombinant protein standards effectively mimic biological complexity for XL-MS experiments.
• Scout outperformed existing software in key performance metrics.

Conclusions:

• The novel XL-MS standards are crucial for the development and validation of proteomic data analysis tools.
• Scout represents a significant advancement in XL-MS data analysis, offering improved accuracy and efficiency.
• This work provides a framework for creating standardized datasets to rigorously evaluate XL-MS software.