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Orbitrap_SciLib
Reputable Mentor II
Reputable Mentor II
Rosa Viner1; David M. Horn1; Eugen Damoc2; Albert Konijnenberg3
IMSC 2018
Introduction: The structural determination of protein complexes plays an important role in the fundamental understanding of their catalytic function. Multiple analytical methods, including hybrid approaches such as cryoelectron microscopy, crosslinking, and native mass spectrometry, are usually required for such type of analysis1. In this study we combined multiple mass spectrometry based structural proteomics techniques to characterize the rabbit 20S proteasome complex. Methods: Rabbit 20S proteasome complex was obtained from Boston Biochem. LC-MS bottom up, crosslinking and intact/top down analysis were performed using a Thermo Scientific™ UltiMate™ 3000 RSLCnano system and a Thermo Scientific™ Orbitrap Fusion™ Lumos™ Mass Spectrometer. Native MS experiments were performed using a Thermo Scientific™ Q Exactive™ UHMR Hybrid Quadrupole-Orbitrap ™ Mass Spectrometer. Data were analyzed with Thermo Scientific™ BioPharma Finder™ 3.0, Thermo Scientific™ Proteome Discoverer™ 2.3, and Thermo Scientific™ ProSightPC™ software. Results: The 20S proteasome complex contains 28 subunits arranged into four stacked rings: seven alpha non catalytic subunits and seven beta-subunits3. In the first series of experiments we performed bottom-up, intact and top-down analysis using databases of proteasome proteins from rabbit, human, and all species in UniProt to create curated fasta database. We then used this curated rabbit 20S proteasome database for all other experiments. For structural characterization, we performed native MS intact and top-down experiments using a Q Exactive UHMR MS. The measured mass of the 28 subunits complex was consistent with expected value of 716 kDa and pseudo- MS3 experiments enabled unambiquous identification of alpha 6 subunit. Crosslinking experiments using DSSO are combined with restraints from native and top-down MS to validate the structure obtained through electron microscopy.


1Thermo Fisher Scientific, San Jose, CA 2Thermo Fisher Scientific, Bremen, Germany 3Thermo Fisher Scientific, Eindhoven, Netherlands
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