The Role of Phosphorylated Residues in Peptide-Peptide Noncovalent Complexes

摘要

Biological mass spectrometry has emerged as an important tool for the study of noncovalent complexes (NCX). One of the main motivations for these studies is the possibility that the structure, stability, and conformations of NCX gas-phase ions may provide information pertaining to their formation in biological systems. Our previous work has highlighted the role of certain amino acid residues, mainly two or more adjacent arginine on one peptide and two or more adjacent glutamate, or aspartate, or a phosphorylated residue on the other in the formation of NCXs between peptides. In this work, we employ mass spectrometry to investigate the gas-phase stability and dissociation pathways of NCXs formed between peptides, through an electrostatic interaction between arginine residues and phosphorylated residues. Two mass spectrometers were used in this study. A Q-TOF Global Ultima mass spectrometer was used for electrospray analysis with a flow rate of 5 (mu)L/min. The mass spectrometer was operated in positive ion mode with a capillary voltage of 3.25 kV, a sampling cone voltage of 50 V, a source temperature of 100 deg C, a desolvation temperature of 300 deg C, a desolvation gas flow rate of 500 L/Hr, and a cone gas flow of 100 L/Hr. For MS/MS analysis, a selection mass window of approx4 Da with a collision gas (Argon) pressure of 13 psi was employed. Collision energies between 3-39 V were used in the collision cell for ion fragmentation. Mass spectra presented are the sum of 50 consecutive 1-second scans. Average intensity values reported in this study are the result of 3 replicate sample runs. A MALDI-TOF/TOF 4700 Proteomics Analyzer was used in this study in both positive and negative ion mode. MS spectra are the sum of 400 laser shots while MS/MS spectra are the sum of 1000 laser shots. For MS/MS analysis, a collision energy of 1 keV with air as the collision gas was used to induce fragmentation.