Identification of Cross-linked Peptides Using Isotopomeric Cross-linkers

Area of Science:

• Biochemistry
• Proteomics
• Chemical Biology

Background:

• Chemical cross-linking combined with mass spectrometry (CL-MS) is vital for protein assembly and interaction studies.
• Identifying cross-linked peptides in complex samples remains a significant challenge.
• MS cleavable cross-linkers improve identification reliability through database searching.

Purpose of the Study:

• Introduce a novel class of MS cleavable cross-linkers: isotopomeric cross-linkers (ICLs).
• Enable confident and efficient identification of cross-linked peptides via whole proteome database searching.
• Present the ICL-MS method for protein interaction analysis.

Main Methods:

• Developed isotopomeric cross-linkers (ICLs) with asymmetric heavy/light isotope incorporation.
• ICLs generate pairs of isotopomeric cross-linked peptides.
• Utilized unique isotopic doublet ions from MS/MS fragmentation for peptide identification and mass determination.

Main Results:

• ICLs facilitate confident identification of cross-linked peptides by generating distinct isotopic doublet ions.
• The ICL-MS workflow allows determination of cross-linked peptide masses from a single MS2 spectrum.
• Demonstrated ICL-MS performance in increasingly complex mixtures using whole proteome database searching.

Conclusions:

• ICLs represent a significant advancement in CL-MS for reliable protein interaction mapping.
• The ICL-MS method enhances the efficiency and confidence of cross-linked peptide identification.
• This approach is particularly valuable for analyzing complex biological samples.