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. 2018 Jan 30;9:80. doi: 10.3389/fmicb.2018.00080

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Copyright © 2018 Panfil, Al-Saleem, Howard, Shkriabai, Kvaratskhelia and Green.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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MS/MS analysis of HBZ ubiquitination sites. HEK293T cells were co-transfected with S-tagged HBZ, FLAG-UB, and FLAG-UBR5 expression vectors. S-tagged HBZ was purified from lysates of transfected HEK293T cells using S beads. The purified HBZ was resolved by SDS-PAGE prior to MS/MS analysis. Representative MS/MS fragmentation spectra are shown for the following two HBZ peptides: (A) DK^∗EEEAVLDGLLSLEEES where lysine 37 is ubiquitinated. Mascot Ion score = 144.5. and (B) K^∗LLQEK^∗EDLMGEVNYWQ where lysines 181 and 186 are ubiquitinated. Mascot Ion score = 52.2. (C) Summary of MS results. The complete HBZ sequence is shown and the protein segments detected by MS/MS analysis are underlined. A schematic is provided to indicate ubiquitination sites on HBZ with respect to individual protein domains.