Novel mass defect labeled chemical crosslinkers and their application to the 34 kDa - Actin biological system

Abstract

In order to improve the identification of crosslinked species within a complex mixture, mass defect label technology can be employed. The improvement is based upon the addition of a mass defect label into the chemical crosslinker of interest. This derivitization changes the mass defect of the species involved in the crosslinking reaction in a manner that increases the identification specificity. In this study, two mass defect labeled chemical crosslinkers were studied, DiBADMPS and DiBBSIAS. The crosslinkers effectiveness at crosslinking, fragmenting and overall identifying the peptides of interest was tested using two small peptides, Neurotensin and Bradykinin, and a small protein/peptide complex, Ribonuelase S. After initial tests were completed, these crosslinkers were applied to the 34 kDa – Actin biological system in order to identify the regions of interactions between these two proteins. To identify the specific amino acids of interest in each protein, HPLC-ESI-FTICR-MS/MS was employed. This allowed for the crosslinked peptides to first be identified and then be fragmented within the ICR cell to produce amino acid sequence information. The results of this study identified the major areas of interactions for 34 kDa protein on Actin.