Signature Ions Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) for Specific and Confident Glycation Site Mapping in Therapeutic Proteins

Wang, Lei; Nwosu, Charles; Gao, Yunfan; Zhu, Mei M.

doi:10.1021/jasms.9b00101

Cited by 6 publications

(2 citation statements)

References 10 publications

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“…To obtain molecular mass information of antimicrobial agents, high-energy collisional dissociation (HCD) of active elution fractions was performed using linear ion trap Orbitrap spectrometer (LTQ Orbitrap XL; Thermo Fisher, United States), a well-established mass spectrometric cleavage technique that produces more fragments and higher quality mass spectra to enhance identification (Wang et al, 2020). The m/z values were measured from 150 to 2,000.…”

Section: Mass Spectrometry Analysis Of Antimicrobial Agentsmentioning

confidence: 99%

Isolation, Identification and Characterization of Two Kinds of Deep-Sea Bacterial Lipopeptides Against Foodborne Pathogens

Zheng

Sun

et al. 2022

Front. Microbiol.

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Under multiple stresses of deep sea, many microorganisms have evolved potentials to produce different metabolites to cope with the stresses they face. In this study, we isolated a bacterial strain Bacillus sp. YJ17 from the deep-sea cold seep. Compared with commercial food preservative nisin, it showed broad and strong antibacterial activities against foodborne pathogens, including multiple resistant bacteria Pseudomonas aeruginosa PAO1 and methicillin-resistant Staphylococcus aureus (MRSA). The active agents were purified by reversed-phase high performance liquid chromatography (RP-HPLC). Analysis of high-energy collision induced dissociation mass spectrometry (HCD-MS) showed that the two active agents belong to family of fengycin and surfactin, and based on results of tandem mass spectrometry (HCD-MS/MS), the amino acid sequence of purified fengycin and surfactin might be Glu-Orn-Tyr-Thr-Glu-Val-Pro-Gln-Tyr-Ile and Glu-Leu/Ile-Leu/Ile-Leu/Ile-Val-Asp-Leu/Ile, respectively. Since the purified fengycin and surfactin exhibited strong inhibition against P. aeruginosa PAO1 and MRSA respectively, the inhibition mechanisms of fengycin against P. aeruginosa PAO1 and surfactin against MRSA were investigated by electron microscopy. After treatment with purified fengycin, the morphology of P. aeruginosa PAO1 became abnormal and aggregated together, and obvious cytoplasmic leakage was observed. After treatment with purified surfactin, the MRSA cells clustered together, and cell surface became rough and jagged. Further study showed that reactive oxygen species (ROS) accumulation and cell membrane damage occurred in P. aeruginosa PAO1 and MRSA after treated with fengycin and surfactin, respectively. Furthermore, typical ROS scavenging enzymes catalase (CAT) and superoxide dismutase (SOD) were also significantly reduced in P. aeruginosa PAO1 and MRSA after treated with fengycin and surfactin, respectively. Therefore, the inhibition mechanisms of fengycin against P. aeruginosa PAO1 and surfactin against MRSA are closely related with accumulation of ROS, which might be due to the decreased activity of CAT and SOD after treated with fengycin and surfactin, respectively. Overall, our study provides good candidates from the deep-sea environment to deal with foodborne pathogens, especially multidrug-resistant bacteria.

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Section: Mass Spectrometry Analysis Of Antimicrobial Agentsmentioning

confidence: 99%

Isolation, Identification and Characterization of Two Kinds of Deep-Sea Bacterial Lipopeptides Against Foodborne Pathogens

Zheng

Sun

et al. 2022

Front. Microbiol.

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“…These diagnostic ions can be generated via different tandem MS techniques. For example, HCD has been used to examine lysine glycation 22 , Selective ETD of N-linked glycopeptides has been performed via HCD derived oxonium ions 23 , 24 , and identification peptides containing lysine modifications has been performed using precursor ion scanning technique with beam-type CID 25 , 26 .…”

Section: Introductionmentioning

confidence: 99%

Diagnostic utility of N-terminal TMPP labels for unambiguous identification of clipped sites in therapeutic proteins

Gunawardena,

Jayatilake,

Brelsford

et al. 2023

Sci Rep

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Protein therapeutics are susceptible to clipping via enzymatic and nonenzymatic mechanisms that create neo-N-termini. Typically, neo-N-termini are identified by chemical derivatization of the N-terminal amine with (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) followed by proteolysis and mass spectrometric analysis. Detection of the TMPP-labeled peptide is achieved by mapping the peptide sequence to the product ion spectrum derived from collisional activation. The site-specific localization of the TMPP tag enables unambiguous determination of the true N-terminus or neo-N-termini. In addition to backbone product ions, TMPP reporter ions at m/z 573, formed via collision-induced dissociation, can be diagnostic for the presence of a processed N-termini. However, reporter ions generated by collision-induced dissociation may be uninformative because of their low abundance. We demonstrate a novel high-throughput LC–MS method for the facile generation of the TMPP reporter ion at m/z 533 and, in some instances m/z 590, upon electron transfer dissociation. We further demonstrate the diagnostic utility of TMPP labeled peptides derived from a total cell lysate shows high degree of specificity towards selective N-terminal labeling over labeling of lysine and tyrosine and highly-diagnostic Receiver Operating Characteristic’s (ROC) of TMPP reporter ions of m/z 533 and m/z 590. The abundant generation of these reporters enables subsequent MS/MS by intensity and m/z-dependent triggering of complementary ion activation modes such as collision-induced dissociation, high-energy collision dissociation, or ultraviolet photo dissociation for subsequent peptide sequencing.

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Efficient HCD-pd-EThcD approach for N-glycan mapping of therapeutic antibodies at intact glycopeptide level

Zhu

Zheng

et al. 2022

Analytica Chimica Acta

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Signature Ions Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) for Specific and Confident Glycation Site Mapping in Therapeutic Proteins

Cited by 6 publications

References 10 publications

Isolation, Identification and Characterization of Two Kinds of Deep-Sea Bacterial Lipopeptides Against Foodborne Pathogens

Isolation, Identification and Characterization of Two Kinds of Deep-Sea Bacterial Lipopeptides Against Foodborne Pathogens

Diagnostic utility of N-terminal TMPP labels for unambiguous identification of clipped sites in therapeutic proteins

Efficient HCD-pd-EThcD approach for N-glycan mapping of therapeutic antibodies at intact glycopeptide level

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