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Structural Analysis of the 20S Proteasome using Native Mass Spectrometry and Ultraviolet Photodissociation

ABSTRACT: The yeast 20S proteasome was analyzed using native mass spectrometry combined with UVPD, HCD, and vT-ESI. Nano-LC-MS-EThcD was used to characterize the primary sequence features of the 20S subunits.

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Jennifer Brodbelt

PROVIDER: PXD056549 | JPOST Repository | Fri Nov 01 00:00:00 GMT 2024

REPOSITORIES: jPOST

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			Action	DRS
	080624_20S_135mMAmAc,15mM-TEAA_HCD_CE=300.RAW	Raw
	080624_20S_135mMAmAc,15mM-TEAA_MS1.RAW	Raw
	080624_20S_135mMAmAc,15mM-TEAA_UVPD=1p1,5mJ_1.RAW	Raw
	080624_20S_135mMAmAc,15mM-TEAA_UVPD=1p1,5mJ_2.RAW	Raw
	080624_20S_135mMAmAc,15mM-TEAA_UVPD=1p1,5mJ_3.RAW	Raw

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Ultraviolet Photodissociation of ESI and MALDI generated protein ions on a Q-Exactive mass spectrometer

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Project description:We analyzed the backbone fragmentation behavior of tryptic peptides of a four protein mixture and of E. coli lysate subjected to Ultraviolet Photodissociation (UVPD) at 213 nm on a commercially available UVPD-equipped tribrid mass spectrometer. We obtained 15,178 high-confidence peptide-spectrum matches by additionally recording a reference beam-type collision-induced dissociation (HCD) spectrum of each precursor. Type a, b and y ions were most prominent in UVPD spectra and median sequence coverage ranged from 5.8% (at 5 ms laser excitation time) to 45.0% (at 100 ms). Overall sequence fragment intensity remained relatively low (median: 0.4% (5 ms) to 16.8% (100 ms) of total intensity) and remaining precursor intensity high. Sequence coverage and sequence fragment intensity ratio correlated with precursor charge density, suggesting that UVPD at 213 nm may suffer from newly formed fragments sticking together due to non-covalent interactions. UVPD fragmentation efficiency therefore might benefit from supplemental activation, as was shown for ETD. Aromatic amino acids, most prominently tryptophan, facilitated UVPD. This points at aromatic tags as possible enhancers of UVPD. Data are available via ProteomeXchange and on spectrumviewer.org/db/UVPD_213nm_trypPep

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Structural Evaluation of Protein/Metal Complexes via Native Electrospray Ultraviolet Photodissociation Mass Spectrometry.

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