Details

Autor(en) / Beteiligte

• Irvine, Gordon W.
• Heinlein, Lina
• Renaud, Justin B.
• Sumarah, Mark W.
• Stillman, Martin J.

Titel

Formation of oxidative and non‐oxidative dimers in metallothioneins: Implications for charge‐state analysis for structural determination

Ist Teil von

• Rapid communications in mass spectrometry, 2017-12, Vol.31 (24), p.2118-2124

Ort / Verlag

England: Wiley Subscription Services, Inc

Erscheinungsjahr

2017

Quelle

Wiley Online Library Journals Frontfile Complete

Beschreibungen/Notizen

• Rationale Metallothioneins (MTs) are a class of dynamic proteins that have been investigated extensively using mass spectrometric methods due to their amenability to ionization. Here we detect the formation of oxidative and non‐oxidative MT dimers using high‐resolution mass spectrometry (HRMS) which has previously been overlooked with lower‐resolution techniques. Methods Recombinant human MT1a and its isolated domain fragments were analyzed by high‐resolution Thermo Q‐Exactive and Bruker time‐of‐flight (TOF) mass spectrometers. Covalent Cys modification was performed using N‐ethylmalemide to probe the effect of Cys oxidation on dimer formation. Results Dimerization was detected in the analysis of select charge states of Zn7MT and apo‐βMT. Specifically, high resolution (140 k) revealed the +6 dimer peaks overlapping with the +3 charge state, but not with the other charge states (+4, +5, +6). The proteins with covalently modified Cys did not show dimer formation in any of their charge states. Apo‐α and apo‐βαMT also did not form dimers under the conditions tested. Conclusions Dimerization of MT was detected for zinc metalated and certain apo‐MT forms with HRMS, which was not seen with lower‐resolution techniques. These dimers appear overlapped only with certain charge states, confounding their analysis for structural characterization of MTs. The Zn‐MT dimers appeared to be non‐oxidative; however, the formation of dimers in the apo‐protein is likely dependent on Cys oxidation.