Collisionally activated dissociation and electron capture dissociation of several mass spectrometry-identifiable chemical cross-linkers.
One of the challenges in protein interaction studies with chemical cross-linking stems from the complexity of intra-, inter-, and dead-end cross-linked peptide mixtures. We have developed new cross-linkers to study protein-protein interactions with mass spectrometry to improve the ability to deal with this complexity. Even the accurate mass capabilities of FTICR-MS alone cannot unambiguously identify cross-linked peptides from cell-labeling experiments due to the complexity of these mixtures resultant from the enormous number of possible cross-linked species. We have developed novel cross-linkers that have unique fragmentation features in the gas phase. The characteristics of these cross-linkers combined with the accurate mass capability of FTICR-MS can help distinguish cross-linking reaction products and assign protein identities. These cross-linkers that we call protein interaction reporters (PIRs) have been constructed with two reactive groups attached through two bonds that can be preferentially cleaved by low-energy CID of the respective protonated precursor ions. After cleavage of the labile bonds, the middle part of the linker serves as a reporter ion to aid identification of cross-linked peptides. This report highlights three new PIRs with new features that have been developed to improve the efficiency of release of reporter ions. The new cross-linkers reported here were tuned with the addition of an affinity tag, a hydrophilic group, a photocleavable group, and new low-energy MS/MS cleavable bonds. This report presents our investigation of the MSMS fragmentation behavior of selected protonated ions of the new compounds. The comprehensive fragmentation of these PIRs and PIR-labeled cross-linked peptides with low-energy collisions and an example of electron capture dissociation in FTICR-MS is presented. These new cross-linkers will contribute to current systems biology research by allowing acquisition of global or large-scale data on protein-protein interactions.
Chowdhury
Saiful M
SM
Department of Chemistry, Washington State University, Pullman, Washington 99164-4630, USA.
Munske
Gerhard R
GR
Tang
Xiaoting
X
Bruce
James E
JE
D003432:Cross-Linking Reagents / Q000737:chemistry*; D017357:Cyclotrons; D004583:Electrons; D005740:Gases; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared
D003432:Cross-Linking Reagents; D005740:Gases; D010446:Peptide Fragments; D011506:Proteins