Mass spectrometry-cleavable cross-linking agents to facilitate structural analysis of proteins and protein complexes, and method of using same

Abstract

Novel cross-linking compounds that can be used in mass spectrometry, tandem mass spectrometry, and multi-stage tandem mass spectrometry to facilitate structural analysis of proteins and protein complexes are provided and have the formula: where X is an N-hydroxy-succinimidyl or similar heterocyclic group. Also provided is a method of mapping protein-protein interactions of protein complexes using various mass spectrometry techniques.

What is claimed is: 
     
         1 . A MS-cleavable cross-linker for proteins and protein complexes, the crosslinker having two symmetric collision-induced dissociation (CID) cleavable sites and the formula: 
       
         
           
           
               
               
           
         
       
       where X is selected from the group consisting of 
       
         
           
           
               
               
           
         
       
       wherein R is methyl or ethyl, 
       and 
       
         
           
           
               
               
           
         
       
     
     
         2 . A MS-cleavable cross-linker as recited in  claim 1 , having the structure: 
       
         
           
           
               
               
           
         
       
     
     
         3 . A method for mapping protein-protein interactions of protein complexes, comprising:
 providing a MS-cleavable cross-linker as recited in  claim 1 ;   forming a cross-linked protein complex by cross-linking proteins with the MS-cleavable cross-linker;   forming protein and/or peptide fragments that are chemically bound to the MS-cleavable cross-linker by digesting the cross-linked protein complex with an enzyme such as trypsin; and   using mass spectrometry (MS) and MS n  to identify the protein and/or peptide fragments.   
     
     
         4 . A method for integrated data analysis workflow for identification of cross-linked peptides, comprising:
 providing cross-linked peptides, each cross-linked peptide comprising an MS-cleavable cross-linker;   performing mass spectrometry on the cross-linked peptides to obtain MS data, MS/MS data, and MS 3  data;   identifying the MS/MS data comprising characteristic fragmentation profiles of MS-cleavable cross-linked peptides to obtain an MS/MS result comprising a list of parent ions corresponding to cross-linked peptide candidates;   mass mapping the MS data using the list of parent ions corresponding to the cross-linked peptide candidates and the MS-cleavable cross-linker against known protein sequences to obtain an MS result;   peptide sequencing the cross-linked peptides using the MS 3  data to obtain an MS 3  result; and   integrating the MS result, the MS/MS result, and MS 3  result to identify at least one of the cross-linked peptides.   
     
     
         5 . The method for integrated data analysis workflow for identification of cross-linked peptides of  claim 4 , wherein the MS-cleavable cross-linker is DSSO. 
     
     
         6 . The method for integrated data analysis workflow for identification of cross-linked peptides of  claim 4 , wherein the MS data is obtained in fourier transform (FT) mode. 
     
     
         7 . The method for integrated data analysis workflow for identification of cross-linked peptides of  claim 4 , wherein the MS/MS data is obtained in fourier transform (FT) mode. 
     
     
         8 . The method for integrated data analysis workflow for identification of cross-linked peptides of  claim 4 , wherein the MS 3  data is obtained using a linear trap quadrupole (LTQ). 
     
     
         9 . The method for integrated data analysis workflow for identification of cross-linked peptides of  claim 4 , wherein the comparing the MS/MS data is carried out using Link-Finder. 
     
     
         10 . The method for integrated data analysis workflow for identification of cross-linked peptides of  claim 4 , wherein the mass mapping is carried out using Protein Prospector MS-Bridge. 
     
     
         11 . The method for integrated data analysis workflow for identification of cross-linked peptides of  claim 4 , wherein the peptide sequencing is carried out using Protein Prospector Batch-Tag. 
     
     
         12 . The method for integrated data analysis workflow for identification of cross-linked peptides of  claim 4 , further comprising reformatting the MS 3  data such that data from MS 3  fragment ions is linked to data from MS/MS parent ions. 
     
     
         13 . The method for integrated data analysis workflow for identification of cross-linked peptides of  claim 4 , wherein the performing mass spectrometry on the cross-linked peptides to obtain the MS data, the MS/MS data, and the MS 3  data comprises:
 obtaining an MS spectrum;   obtaining an MS/MS spectrum;   obtaining an MS 3  spectrum;   extracting the MS data from the MS spectrum;   extracting the MS/MS data from MS/MS spectrum; and   extracting the MS 3  data from the MS 3  spectrum.   
     
     
         14 . The method for integrated data analysis workflow for identification of cross-linked peptides of  claim 4 , wherein the MS-cleavable cross-linker has the formula: 
       
         
           
           
               
               
           
         
       
       where X is selected from the group consisting of 
       
         
           
           
               
               
           
         
       
       wherein R is methyl or ethyl, 
       and