[Vacuum ultraviolet laser dissociation and proteomic analysis of halogenated peptides]

Chemical modifications are widely used in research fields such as quantitative proteomics and interaction analyses. Chemical-modification targets can be roughly divided into four categories, including those that integrate isotope labels for quantification purposes, probe the structures of proteins through covalent labeling or cross-linking, incorporate labels to improve the ionization or dissociation of characteristic peptides in complex mixtures, and affinity-enrich various poorly abundant protein translational modifications (PTMs). A chemical modification reaction needs to be simple and efficient for use in proteomics analysis, and should be performed without any complicated process for preparing the labeling reagent. High reaction specificity, which reduces product complexity, and mild biocompatible reaction conditions are also favored. In addition, modification labels should be compatible with mass spectrometry to prevent interference from ionization and dissociation processes. Pulsed ultraviolet (UV) lasers can produce large amounts of active radical species within a few nanoseconds for use in rapid photochemical-modification processes. Usually, UV lasers with wavelengths greater than 240 nm are used in current in-situ photochemical-modification methods; consequently, special conjugated photoreaction probes need to be designed and oxidants and catalysts added, which reduce the biocompatibility of the reaction. The high single-photon energy of the 193 nm laser is capable of efficiently exciting conventional photo-inert substances in aqueous solution, leading to efficient photochemical peptide modifications. In this study, we developed a new method for photochemically brominating and iodinating enzymatic protein samples extracted from complex tissue with a 193 nm ArF nanosecond pulsed laser, which efficiently brominated tyrosine, histidine, and tryptophan, and iodinated tyrosine and histidine. Tandem mass spectrometry (MS/MS) can generate fragmentation patterns of ions which can afford diagnostic molecular fingerprints to decipher sequences of biopolymers such as peptides. Peptide fragmentation is commonly implemented using collision-based, electron-based, or photodissociation-based methods. Compared with the most commonly used collision-based methods, ultraviolet photodissociation (UVPD) uses high-energy ultraviolet photons with wavelengths shorter than 200 nm to excite and dissociate ions. Single-pulse excitation can provide the energy required to promote ions into their excited electronic states, with excitation speeds of up to several nanoseconds. Since dissociation may occur directly from the excited states, UVPD spectra can show a wide variety of fragmentation pathways, thereby providing more sequence and structural information. The most commonly used wavelengths are 157, 193, and 266 nm. UVPD has been integrated into high-resolution orbitrap mass spectrometer by adding optical windows and other optics to direct the photons to the analyte ions, and by implementing a triggering method that synchronizes the photoirradiation process with ion-analysis events. The large photoabsorption cross sections of peptides at 193 nm and the resulting high internal energy deposition can generate abundant fragment ions and achieve high sequence coverage. The excellent fragmentation performance offered by 193 nm UVPD of peptides with its high sequence coverage and lack of charge-state dependence, has motivated its use in high-throughput proteomics. Photochemically brominated and iodinated mouse-liver tryptic peptides were further characterized by 193 nm UVPD tandem mass spectrometry with the aim of analyzing their sequences, modification sites, and photodissociation mechanisms. Br and I atoms strongly absorb 193 nm photons; consequently, UVPD can cleave C-Br/C-I bonds at halogenated sites to generate peptide radical ions, with further peptide-backbone fragmentation caused by radical migration. In addition, the combination of 193 nm UVPD with conventional high-energy collision-induced dissociation (HCD) mode improves the identification-reliability of halogenation sites in proteomics. Therefore, integrating photochemical halogenation and 193 nm UVPD can trigger novel radical-dissociation pathways, thereby improving analytical proteomics performance.