| Modern mass spectrometry (MS) technology has several distinct advantages, including high sensitivity, accuracy, through-put, automation, etc, which has been applied to most of life science research fields, such as proteomics, neurochemistry, physiology, nucleic acidic chemistry, cell signaling, etc. This work focuses on establishment of comprehensive mass spectrometry and related technologies to analyze six peptide families with complicated structures, involving antitumor marine cyclic peptides, linear precursor of cyclic peptides, disulfide-linked peptides, large neuropeptides, racemized peptides, proteolytic peptides, and aims to develop novel MS-based methodology for peptide mapping. In addition, the peptide fragmentation mechanism in MS was also studied in deep, and several related research works were introduced including synthesis of cyclic peptides, preparation of cross-linked enzyme aggregates, retention mechanism of reversed-phase chromatography, physiology test of neuropeptides, etc.Comprehensive MS is a useful tool box that contains protocols of sample pretreatment and separation, techniques of ionization source, ion selection, and ion analyzer, strategies of data analysis, peptide sequencing programming, and database searching, theories of gas-phase ion chemistry, enzymology, matrix chemistry, MS imaging, which was combined and further used to characterization of peptides and biological functions. The research details are described as following,First, the multistage MS method was established for sequencing of antitumor marine cyclic peptides. Based on bx→bx-1 fragmentation pathway, the b ion can dissociate to lose one C-terminal amino acid residue in every stage of CAD, producing two sets of b ions. The cyclic peptide sequence can be determined by observation of these b ions. The semiempirical calculation was used to investigate the normal fragmentation and abnormal rearrangement pathways. The selective ring opening of cyclic peptides was studied, and Asn-induced ring opening mechanism was proposed.Second, during characterization of some peptides in an ion trap, it was noted that many internal amino-acid residues could be lost from singly charged b ions. The unique fragmentation consisting of multiple steps is induced by a cyclization reaction of b ions, the mechanism of which has been probed by experiments of N-acetylation, MSn, rearranged-ion design and activation-time adjustment. The fragmentation of synthetic cyclic peptides demonstrates that a cyclic peptide intermediate (CPI) formed by b ion cyclization exhibits the same fragmentation pattern as a protonated cyclic peptide. Although no rules for the cyclization reaction were discerned in the experiments of peptide modification, the fragmentations of a number of b ions indicate that the"Pro and Asn/Gln effects"can influence ring openings of CPIs. In addition, large-scale losses of internal residues from different positions of a-type ions have been observed. The fragmentation is initiated by a cyclization reaction forming an a-type ion CPI. This CPI with a fixed-charge structure cannot be influenced by the"Pro effect", causing a selective ring opening at the amide bond Pro-Xxx rather than Xxx-Pro.Third, using 1,5-diaminonaphthalene (DAN) as a reductive screening matrix for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric profiling of disulfide bond-containing C-type allatostatin peptides, we identified and sequenced a novel C-type allatostatin peptide (CbAST-C1), present in the pericardial organs of the crab, Cancer borealis. Electrophysiological experiments demonstrated that the AST peptides inhibited the frequency of the pyloric rhythm of the STG, in a state-dependent manner. At 10-6M, both peptides were only modestly effective when initial frequencies of the pyloric rhythm were >0.8 Hz, but almost completely suppressed the pyloric rhythm when applied to preparations with starting frequencies < 0.7Hz.Forth, the multifaceted MS method was established for characterization of CHH-family peptides in nervous system of blue crab. The peptides SG-CHH, SG-MIH, SG-CPRP, SG-tCPRP and SG-PPRP were identified in pericardial organ, and the peptides PO-CHH, PO-CPR and PO-tCPRP were identified in sinus gland.The isotopic labeling strategy and MS imaging strategy were established for quantitation and distribution analysis of CHH-family peptides and homologous large peptides, which laid the foundation for future study on physiological function. Fifth, the retention characteristic of hydrophilic heptapeptide on RP-HPLC was discussed, and the process of'pre-adsorption'was proposed in order to take full advantage of contributions of both retention mechanisms of partition and adsorption to separation. Thus by this method four ideal RP-HPLC peaks were detected. Meanwhile, the MS information indicated that the four peaks had the same value of m/z 761.5[M + H]+. Then through simultaneous peptide sequencing of each racemization products and figure superposition of tandem MS (MSn), the two sets of segment ions, b and y, were observed except b1 and y1, which showed that the four racemization products of synthetic heptapeptide were successfully identified.Sixth, immobilized enzymes have been utilized in proteomics due to several distinct advantages. However, most immobilization methods require an inactive medium to support the enzyme, which occupies 90–99% enzymatic volume, lowering effective enzyme concentration. Herein, we introduce a carrier-free enzyme immobilization technology into proteomics for rapid proteolysis. Cross-linked trypsin aggregates (CLEA-trypsin) were prepared by a simple and low-cost method, showing excellent proteolysis efficiency, improved thermal stability, and reduced enzyme autolysis. A high throughput proteomic strategy was developed by incorporating on-plate CLEA-tryptic digestion and high-accurate MALDI-FTMS peptide mass fingerprinting, and an average sequence coverage of 46% was obtained by digestion of seven protein standards for 5 min at 77°C.
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