Analysis for disulfide bonds in polypeptide sequences based on ultraviolet photodissociation combined with linear ion trap mass spectrometry

Abstract

The analysis of dissociation products of disulfide bonds in peptide sequences has been increasingly applied in various fields. Ion dissociation is typically performed by tandem mass spectrometry, in which the overall molecular weight is determined during the first stage and the protein sequence and structure are obtained by dissociation during the second stage. However, conventional dissociation methods have some disadvantages, such as insufficient dissociation efficiency, and low product signals and varieties. Ultraviolet photodissociation (UVPD) is a rapid, efficient, and practical dissociation method for protein analysis, which excites the biomolecule skeleton and then directly excites the molecules, forming the excited states within microseconds. Although the development of mass spectrometer platforms coupled with UVPD has been reported to date, most of these studies have focused on the application of UVPD in mass spectrometry (MS). Discussions on the experimental setup for UVPD lasers, including the trigger process of the ultraviolet (UV) laser, the timing sequence of the entire dissociation and detection process, and their influence on dissociation, are limited. Therefore, an instrument platform was developed with a linear ion-trap mass spectrometer and an excimer laser to achieve UVPD by adjusting the laser trigger signal. The platform dissociated the disulfide bond of the amino acid sequence ARACAKA-ECG into two characteristic valence states (m/z = 498 and 332), and their highest intensities were obtained by optimizing the experimental conditions of the platform. The dissociation results of UVPD showed the characteristic peaks of ARACAKA (m/z 345) that were produced by disulfide bond cleavage, but these peaks were not detected by collision-induced dissociation (CID). Therefore, it was confirmed that the developed platform realized UVPD of the disulfide bond of the amino acid sequence of the polypeptide. Moreover, the developed platform provides a reference for the implementation of UVPD for peptide sequencing.