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Identification and High-Resolution Imaging of α-Tocopherol from Human Cells to Whole Animals by TOF-SIMS Tandem Mass Spectrometry.

ABSTRACT: A unique method for identification of biomolecular components in different biological specimens, while preserving the capability for high speed 2D and 3D molecular imaging, is employed to investigate cellular response to oxidative stress. The employed method enables observing the distribution of the antioxidant α-tocopherol and other molecules in cellular structures via time-of-flight secondary ion mass spectrometry (TOF-SIMS (MS¹)) imaging in parallel with tandem mass spectrometry (MS²) imaging, collected simultaneously. The described method is employed to examine a network formed by neuronal cells differentiated from human induced pluripotent stem cells (iPSCs), a model for investigating human neurons in vitro. The antioxidant α-tocopherol is identified in situ within different cellular layers utilizing a 3D TOF-SIMS tandem MS imaging analysis. As oxidative stress also plays an important role in mediating inflammation, the study was expanded to whole body tissue sections of M. marinum-infected zebrafish, a model organism for tuberculosis. The TOF-SIMS tandem MS imaging results reveal an increased presence of α-tocopherol in response to the pathogen. Graphical Abstract ᅟ.

SUBMITTER: Bruinen AL

PROVIDER: S-EPMC6060986 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Canine oral tumors are relatively common neoplasms in dogs. For disease monitoring and early diagnosis, salivary biomarkers are appropriate because saliva collection is non-invasive and requires no professional skills. In the era of omics, matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF MS) coupled with liquid chromatography-tandem MS (LC-MS/MS) are suitable to identify potential disease-associated peptides and proteins. The present study aimed to use MALDI-TOF MS and LC-MS/MS to search for particular peptide mass fingerprints (PMFs) and conceivable biomarkers in saliva of dogs with early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC), benign oral tumors (BN), and periodontitis and healthy controls (CP). Pooled saliva samples in each group were used to be representative of population change. Unique PMFs were obtained and specific peptide fragments were sequenced by LC-MS/MS and BLAST-searched with mammalian protein databases. Seven peptide fragments appeared in the tumor groups (EOM, LOM, OSCC and BN) at 1096, 1208, 1322, 1794, 1864, 2354 and 2483 Da, two peptide fragments appeared in the LOM and OSCC groups at 2450 and 3492 Da, and in the CP controls at 2544 and 3026 Da. Also, protein-chemotherapy drug interaction networks were exhibited. Using western blot analysis, the expression of sentrin-specific protease 7 (SENP7), a peptide fragment at 1096 Da, in OSCC was significantly increased, as was the expression of TLR4, a peptide fragment at 3492 Da, in LOM and OSCC, compared with the CP group. The expression of nuclear factor kappa B (NF-κB), a TLR4 partner, was notably increased in OSCC compared with CP, BN and EOM. The expression was also enhanced in LOM compared with EOM. Expressed protein sequences from western blots were verified by LC-MS/MS. Western blots were then performed with individual samples in each group. The results showed the elevated expression of TLR4 in LOM and OSCC, compared with that in CP and BN, the increased expression of NF-κB in LOM and OSCC, compared with CP and in LOM compared with BN, and the enhanced expression of SENP7 in LOM and OSCC, compared with that in CP and BN. In conclusion, discrete clusters of EOM, LOM, OSCC, BN and CP groups and potential protein candidates associated with the diseases were demonstrated by salivary proteomics. Western blot analysis verified SENP7, TLR4 and NF-κB as potential salivary biomarkers of canine oral tumors.

Dataset Information

Identification and High-Resolution Imaging of α-Tocopherol from Human Cells to Whole Animals by TOF-SIMS Tandem Mass Spectrometry.

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