Collision-Induced Dissociative Crosslinking Reagents and Methodology for the Structural Analysis of Proteins and Protein-Protein Interactions using Tandem Mass Spectrometry.

protein structure;crosslink;mass spectrometry;Proteomics

Soderblom, Erik James ; Dr. Steven D. Clouse, Committee Member,Dr. Michael B. Goshe, Committee Chair,Dr. John Cavanagh, Committee Member,Dr. Dennis T. Brown, Committee Member,Dr. Carla Mattos, Committee Member,Soderblom, Erik James ; Dr. Steven D. Clouse ; Committee Member ; Dr. Michael B. Goshe ; Committee Chair ; Dr. John Cavanagh ; Committee Member ; Dr. Dennis T. Brown ; Committee Member ; Dr. Carla Mattos ; Committee Member

University:North Carolina State University

关键词: protein structure;    crosslink;    mass spectrometry;    Proteomics;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/4064/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

Chemical crosslinking combined with mass spectrometry is a viable approach to study the low-resolution structure of proteins as well as the topological organization of protein-protein complexes.The identification of the residues involved in the crosslink, however, remains analytically challenging due to the nature of the crosslink itself and its influence on the resulting mass spectrum.To address these challenges, we have developed a novel class of collision-induced dissociative chemical crosslinking reagents which incorporate a gas-phase cleavable aspartyl-prolyl bond into the linker region of the reagent.The specific dissociation of the crosslinked complex allows for a tandem mass spectrometry analysis to be performed on each of the individual peptides involved in the crosslink and therefore simplifies the analysis of the crosslinked products.Following a description of the organic synthesis and purification protocols developed for two collision-induced dissociative crosslinking reagents, a systematic dissociation study of various lysine containing polyalanine peptides was conducted to determine optimized energies for reagent dissociation.Once optimal conditions had been established, methodologies for incorporating the novel crosslinking reagents into automated high-performance liquid chromatography-tandem mass spectrometry analysis were developed on various mass spectrometry platforms and validated with several different model protein systems.To apply our novel crosslinking reagent and methodology to a structurally unknown protein system, a complete crosslinking study of the transition-state regulator AbrB from Bacillus subtilis was conducted.The structural constraints obtained from these crosslinking studies were used to generate a three-dimensional model of full length AbrB which will serve as a platform for additional biochemical studies.