Study On Noncovalent Interaction Of Bradykinin Peptide Fragment By Mass Spectrometry

In recent years, great progress has been made in the studies of gas-phase peptides and proteins structure as the widely use of mass spectrometry technology in life science field. Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) and other "soft" ionization mass spectrometry technologies, were not only used to detect the heat-labile, polar, non-covalent compounds, but also allow complex completely enter the gas phase from solution phase to be detected directly under certain condition. Thus we could obtain information of the stoichiometry of molecular interactions, combined with the energy situation and the information of energy under near physiological state, so that the application of mass spectrometry has been extended from small molecules to large biological molecules. Therefore, electrospray mass spectrometry (ESI-MS) has been a powerful tool for investigating non-covalent interactions in proteins or peptides.In this dissertation, we applied ESI-MS as the main technology for investigating the non-covalent interaction and hydrogen bond between peptides of Bradykinin, the fragments of non-covalent complexes and the non-covalent interactions between enclosed peptides.First we explore the non-covalent interaction between peptides of a Bradykinin molecule (R1P2P3G4F5S6P7F8R9), several peptides which have the same amino acid sequence were synthesized. In the first fracture mode, we synthesized two peptides RS-6(RPPGFS) and PR-3(PFR) according to our requirement; in the second fracture mode, two peptides RF-5(RPPGF) and SR-4(SPFR) were obtained. By removing the arginine at N-terminal or C-terminal, we designed and synthesized another four kinds of polypeptide. In our experiments, any two kinds of these eight polypeptides were mixed in a molar ratio of1:1, respectively. In fracture mode1, when R9was removed, the peptide PF did not bind to any other fragment peptides. While in fracture mode2, the non-covalent binding still took place between fragment peptides, whether or not R1or R9was removed, which indicates that serine is likely to be in the position of β-turn.For the complexes of RPPGFS with PFR, and the complexes of RPPGF with SPFR, the binding constant Kst values determined by mass spectrometric titrations were3.53x103and3.16×103respectively, which are greater than the Kst value (1.25×103) for the complexes of PPGF with SPF. The non-covalent binding between two peptides was further confirmed by collision-induced dissociation (CID) in a tandem mass spectrometer. It is demonstrated that the binding strength of RPPGFS and PFR, RPPGF and SPFR complexes is much stronger than other peptide complexes. The non-covalent binding between peptides without R1and R9is much weaker. The fragments of complexes of RPPGFS and PFR, RPPGF and SPFR at50eV are almost b-y ions. At the same time, we got neutral loss of water molecule(H2O,18), formaldehyde (CH2O,30), or the guanidino from the arginine (CN2H2,42)、(CN3H5,59)。These results are in accordance with the conformation of Bradykinin.The third part, we use ESI-MS to study the non-covalent interactions between peptides which were enclosed with amino-terminal modification and carboxyl-terminal modification. ESI-MS results revealed that it is harder for these peptides to form non-covalent complexes. That’s because the enclose peptides are more stable and the side chains are difficult to form hydrogen bond with other side chains of another peptide. CID results showed that, most non-covalent complexes start to fragment into the corresponding peptides at20eV, few peptides at25eV. In the energy range of30eV-45eV, the peak intensity of the complexes could fall down below10%of the strongest peak intensity.