Mass spectrometry identifiable cross-linking strategy for studying protein-protein interactions.
A new mass spectrometry identifiable cross-linking strategy has been developed to study protein-protein interactions. The new cross-linker was designed to have two low-energy MS/MS-cleavable bonds in the spacer chain to provide three primary benefits: First, a reporter tag can be released from cross-link due to cleavage of the two labile bonds in the spacer chain. Second, a relatively simple MS/MS spectrum can be generated owing to favorable cleavage of labile bonds. And finally, the cross-linked peptide chains are dissociated from each other, and each then can be fragmented separately to get sequence information. Therefore, this novel type of cross-linker was named protein interaction reporter (PIR). To this end, two RINK groups were utilized to make our first-generation cross-linker using solid-phase peptide synthesis chemistry. The RINK group contains a bond more labile than peptide bonds during low-energy activation. The new cross-linker was applied to cross-link ribonuclease S (RNase S), a noncovalent complex of S-peptide and S-protein. The results demonstrated that the new cross-linker effectively reacted with RNase S to generate various types of cross-linked products. More importantly, the cross-linked peptides successfully released reporter ions during selective MS/MS conditions, and the dissociated peptide chains remained intact during MS(2), thus enabling MS(3) to be performed subsequently. In addition, dead-end, intra-, and inter-cross-linked peptides can be distinguished by analyzing MS/MS spectra.
Tang
Xiaoting
X
Department of Chemistry, Washington State University, Pullman, WA 99164-4630, USA.
Munske
Gerhard R
GR
Siems
William F
WF
Bruce
James E
JE
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping / Q000379:methods*; D012260:Ribonucleases / Q000737:chemistry
D003432:Cross-Linking Reagents; D012260:Ribonucleases; C022542:ribonuclease S