Author of

• 25940

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  P2.03 - Biology (Not CME Accredited Session) (ID 952)

  • Event: WCLC 2018
  • Type: Poster Viewing in the Exhibit Hall
  • Track:
  • Presentations: 1
  • Moderators:
  • Coordinates: 9/25/2018, 16:45 - 18:00, Exhibit Hall

  • 26949

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    P2.03-13 - SWATH MS Analysis of Serine Hydrolase Activity in Human Lung Adenocarcinoma for Biomarker Discovery (ID 13300)

    16:45 - 18:00  |  Author(s): Stephan Arni
    • Abstract

    Background
    

    Serine hydrolases (SHs), one of the largest enzyme families, have previously been shown to be implicated in the development of lung cancers. Activity-based protein profiling (ABPP) is a proteomic method that uses active site-directed chemical probes to selectively target the active form of the subsets of the enzymes in question, and then by a combination of a streptavidin-biotin enrichment step and mass spectrometry quantifies the catalytically active amount of the enzyme molecule. In this project, we monitored both forms of serine hydrolase, the “catalytically active” and “inactive”, in a distinct patient cohort of lung adenocarcinoma biopsies. For this purpose, we combined the activity-based proteomics for serine hydrolase and SWATH mass spectrometry (MS), which ensures highly reproducible protein quantification in a large panel of clinical samples.

    a9ded1e5ce5d75814730bb4caaf49419 Method
    

    Twenty four lung biopsies of long- and short surviving patients with stage IIIA adenocarcinomas and their normal tissue counterparts were available as OCT-embedded tissue. The common OCT sample clean-up by organic solvents is not compatible with the ABPP protocol since organic solvents can inactivate the molecular structure of the enzymes. The additional challenge of the ABPP protocol includes a streptavidin-biotin enrichment step of the active enzymes, which also leads to sample contamination by streptavidin peptides, and negatively influences MS spectra analysis and, consequently, biomarker discovery.

    Therefore, we proceeded with optimizations of sample preparation in ABPP experiments and developed an OCT clean-up protocol compatible with “enzyme-substrate binding” of activity-based chemical probes and the targeted portion of the active enzyme. To prevent digestion on streptavidin beads and MS spectra contamination, we used the reproducible and accurate SWATH MS to indirectly measure the active enzyme form in the biopsy-extract solution.

    4c3880bb027f159e801041b1021e88e8 Result
    

    We identified over 4000 lung tissue proteins from a few milligrams of OCT-embedded biopsies, and confirmed good data quality for further SH enzyme quantification. In addition to the analysis of total proteome, 278 distinct proteins were identified on the parallel streptavidin bead samples, with chemical probes being used to deplete the active enzyme from the biopsy-extract.

    Each SWATH experiment reported the percentage of “active” enzyme form through the indirectly measured ratio between the “inactive SHs” (sample depleted for active SHs) and the “total” SHs (non-depleted sample). We detected around 80 enzymes with the “active” form comparably measured in three independent experiments, which amount generally accounted for between 5–55% of the total enzyme concentration.

    8eea62084ca7e541d918e823422bd82e Conclusion
    

    Combination of ABPP and SWATH MS enables highly reproducible protein quantification in biomarker discovery.

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