Horse heart myoglobin

HEWL, penta-N-acetylchitopentaose (NAG₅), dithiotreithol (DTT), iodoacetamide, calmodulin, equine cytochrome c, horse heart myoglobin, melittin and ubiquitin were purchased from Sigma-Aldrich (Poole, UK). Trypsin and AspN were obtained from Promega (Southhampton, UK). Lys48 poly-ubiquitin ladder, wild-type USP5 and Lys48 diubiquitin were purchased from Boston Biochem (Cambridge, MA, USA). C335A USP5 was expressed and purified as previously described34 (link). Photoleucine 1 was purchased from Thermo Fisher Scientific, Loughborough, UK, and aryldiazirine 2 synthesized as described above.

Urine samples were diluted in formic acid (0.1% (v/v) in HPLC grade water) to a protein concentration of approximately 5 pmol/μl. The samples were injected onto a C4 desalting trap (Waters MassPREP™ Micro desalting column, 2.1 × 5 mm, 20 μm particle size, 1000 Å pore size) (Waters, Manchester, UK) that was fitted on a Waters nano ACQUITY Ultra Performance liquid chromatography^® (UPLC^®) system. The chromatography system was coupled to a Waters SYNAPT™ G1 QTof mass spectrometer fitted with an electrospray source. Protein was eluted over a 10 min acetonitrile (ACN) gradient (5–95% (v/v)) at 40 μl/min. Data were collected between 500–3500 m/z. The data were processed using maximum entropy deconvolution (MAX ENT 1, Mass Lynx version 4.1, Waters) at 0.5 Da/channel over a mass range of 8500–10000 Da. The mass spectrometer was calibrated externally with horse heart myoglobin (1 pmol/μl, Sigma).

ApoMb was prepared from horse-heart myoglobin (Sigma-Aldrich) following the butanone method to extract the heme group (as performed in^{50 (link)}), adapting the method described in^{60 (link)}), and then refolded by dialysis in 20 mM NaH₂PO₄/Na₂HPO₄ (Sigma Life Science, >99.5% and Sigma-Aldrich, >99%) pH 7 buffer and distilled water. Before storage in the freezer, the apo-Mb solution was lyophilized. To replace the exchangeable protons by deuterium ions, the freeze-dried apo-Mb powder was dissolved in heavy water (99.9% ²H, Sigma-Aldrich), incubated for 1 day, and lyophilized again. The obtained powder was stored at −20 °C. In order to obtain the molten globule state of apoMb the powder was dissolved in ²H₂O and centrifuged to remove the large aggregates. In the supernate solution of concentration 2 mg/mL and pH 6, ²HCl 0.1 M (Sigma-Aldrich) was added until the pH-read out value was 3.6 (monitored by pH meter Methrom). This corresponds to a a pD value of 4. The buffer exchanged protein solution was centrifuged (Heracus Instruments) to the final concentrations (Vivaspin 3,000 MWCO concentration units, Sartorius, Göttingen, Germany).

Horse heart myoglobin was purchased from Sigma-Aldrich. Hypericin was obtained from HWI Analytik GmbH. All were used as received.