Cross-linking compositions and related methods of isotope tagging of interacting proteins and analysis of protein interactions
View Patent ↗An isotope labeled asymmetric cross-linker is provided for the detection of cross-linked peptides. A cross-linking and mass spectrometry strategy, referred to as isotope tagging of interacting proteins (iTIP), improves the specificity of detecting cross-linked peptides and accurate identification of the interacting peptide sequences via the incorporation of isotopic signatures that are readily observed in the MS/MS spectrum. Isotope tagged peptides can be identified using mass spectrometry based on doublet peaks in a spectrum. Spectra can be subjected to database search strategies available for the sequencing of linear or non-cross-linked peptides.
1. An isotope labeled amine reactive cross-linker, comprising:
a 3,3′-dithiobis(sulfosuccinimidyl sulfo propionate) molecule comprising a disulfide bond; and
a deuterium label positioned on one side of the disulfide bond of the 3,3′-dithiobis(sulfosuccinimidyl sulfo propionate) molecule, wherein the deuterium label causes the 3,3′-dithiobis(sulfosuccinimidyl sulfo propionate) molecule to be structurally asymmetrical across the disulfide bond, wherein the disulfide bond can be dissociated, wherein dissociation of the disulfide bond creates a first and a second cleavage product, wherein one of the first or second cleavage products comprises the deuterium label, wherein the first and a second cleavage products differ in mass, and wherein the nominal mass of the cleavage products differs by 4 Daltons.
2. The cross-linker of claim 1 , wherein the deuterium label is on a methylene carbon of the 3,3′-dithiobis(sulfosuccinimidyl sulfo propionate) molecule.
3. The cross-linker of claim 1 , comprising the following chemical structure:
wherein b is the disulfide bond, wherein the a-b segment is a protonated light tag that can attach to NH 2 of a lysine residue or protein N-terminus during cross-linking, wherein the b-c segment is a deuterated heavy tag that can attach to NH 2 of a lysine residue or protein N-terminus during cross-linking.
4. An isotope labeled amine reactive cross-linker, comprising:
a 3,3′-dithiobis(sulfosuccinimidyl sulfo propionate) molecule comprising a disulfide bond; and
a deuterium label positioned on one side of the disulfide bond of the 3,3′-dithiobis(sulfosuccinimidyl sulfo propionate) molecule, wherein the deuterium label causes the 3,3′-dithiobis(sulfosuccinimidyl sulfo propionate) molecule to be structurally asymmetrical across the disulfide bond,
wherein the cross-linker comprises the following chemical structure:
wherein b is the disulfide bond, wherein the a-b segment is a protonated light tag that can attach to NH 2 of a lysine residue or protein N-terminus during cross-linking, wherein the b-c segment is a deuterated heavy tag that can attach to NH 2 of a lysine residue or protein N-terminus during cross-linking.