Structural analysis of multiprotein complexes by cross-linking, mass spectrometry, and database searching

Area of Science:

• Biochemistry
• Structural Biology
• Bioinformatics

Background:

• High-resolution structural analysis of protein complexes is often limited.
• Large protein complexes with coiled-coil structures pose significant challenges for traditional methods.

Purpose of the Study:

• To develop and validate a novel computational approach for analyzing cross-linking mass spectrometry data.
• To determine the structural organization of the NDEL1 homodimer and the NDC80 heterotetramer.

Main Methods:

• Combined cross-linking, mass spectrometry, and bioinformatics.
• Development of a purpose-built database for matching spectra with cross-linked peptides.
• Scoring system for identification quality and false positive rate estimation.

Main Results:

• Successfully elucidated structural parameters of the NDEL1 homodimer and NDC80 heterotetramer.
• Determined the directionality of the NDEL1 homodimeric coiled coil.
• Resolved the register of NDC80 heterodimeric coiled coils and its tetramerization region organization.

Conclusions:

• The novel integrated approach overcomes data analysis bottlenecks in cross-linking mass spectrometry.
• This method is particularly effective for studying protein complexes intractable by standard structural techniques.
• Provides critical insights into the architecture of large, complex protein assemblies.