Real-Time Library Search Increases Cross-Link Identification Depth across All Levels of Sample Complexity

Area of Science:

• Biochemistry
• Structural Biology
• Proteomics

Background:

• Cross-linking mass spectrometry (XL-MS) is vital for studying protein structure and interactions.
• Identifying cross-linked peptides is challenging due to their low abundance and interference from mono-links.
• Current methods struggle to differentiate chemically similar cross-links and mono-links.

Purpose of the Study:

• To develop a method for distinguishing cross-links from mono-links in XL-MS.
• To improve the efficiency and accuracy of cross-link identification.
• To enhance high-throughput XL-MS applications.

Main Methods:

• Utilized PhoX/tBu-PhoX cross-linkers and analyzed intensity ratios of four diagnostic peaks.
• Implemented real-time library search (RTLS) for targeted MS2 scan triggering.
• Applied the method to single protein samples and intact E. coli cells.

Main Results:

• Established partial distinction between cross-links and mono-links based on diagnostic peak intensity ratios.
• RTLS significantly increased the number of identified cross-links.
• Demonstrated improved cross-link identification in unenriched samples and short gradients.

Conclusions:

• The RTLS strategy effectively enhances cross-link identification in XL-MS.
• This approach offers advantages for high-throughput studies and limited sample/instrument time.
• The findings advance the utility of XL-MS for structural and interaction analysis.