Asymmetric Chemical Cross-Linking Enables Isotope Tagging of Interacting Proteins (iTIP)

Author(s): Harsha P Gunawardena

An isotope labeled cross-linker (asymmetric d4-DTSSP) was developed to streamline the efforts needed for the detection of cross-linked peptides. The cross-linking and mass spectrometry strategy we call Isotope Tagging of Interacting Proteins (iTIP) has improved the specificity of detecting cross-linked peptides and the correct identification of the interacting peptide sequences via the incorporation of isotopic signatures that are readily observed in the MS/MS spectra. All tryptic peptides derived from the cross-linking reactions of a protein complex are subjected to ETD-MS2 which results in the facile cleavage of the cross-linker at the disulfide bond and the release of inter-linked polypeptide chains that are detected as a pair of peaks (doublets) in the MS2 spectrum. The constituent peptide halves that are tagged by the heavy/light ends of the cross-linker are easily mass-selected from all other fragment ions, and each polypeptide half is then subjected to CID- or HCD-MS3 for identification. The MS3 spectra are subjected to conventional database search strategies available for the sequencing of linear or non-cross-linked peptides. The confident identification of each polypeptide is further aided by the presence of a stable isotope labeled fragment ions that localizes the cross-linked site on the polypeptide sequence.

© 2016-2024, Copyrights Fortune Journals. All Rights Reserved