Platinum coated emitter

Approximately 4 μL of incubated sample buffer exchanged into 200 mM ammonium acetate (pH = 7.4) was loaded in a platinum-coated emitter (Thermo Scientific, Waltham, MA) and electrosprayed directly into the mass spectrometer via a nanoESI source. An Exactive Plus EMR Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA) was used to obtain the native mass spectrum of the complex with the following settings: capillary voltage of 1.5 kV, capillary temperatures of 100 °C and 50 °C, in-source CID of 10 V, and HCD collision energy of 10 V. The AGC target was set to 5 e⁶. The mass resolving power was 140,000 at m/z = 200. The spectra were dependent on instrumental tune conditions, and the conditions for data in Figure 2A were adjusted to show minimal fragmentation, which is desired. Data processing was done by using Thermo Xcaliber Qual Browser 4.0.

ISCID and ECD product-ion mass spectra were acquired on a Bruker Scientific 12 T FT-ICR mass spectrometer (Bruker, Bremen, Germany) using 4 μL of incubated sample buffer exchanged into 100 mM EDDA (pH = 7.4), loaded into a platinum-coated emitter (Thermo Scientific, Waltham, MA), and infused directly into the mass spectrometer via a nanoESI source. The source settings were capillary voltage 1.5 kV, drying gas temperature 100 °C, and drying gas flow rate 1.5 L/min. Skimmer I voltage was varied to pre-activate ions, and the downstream collision voltage was optimized for subsequent collisional activation. Other ion-optics parameters were adjusted to optimize signal intensities. Ion accumulation time was 3 s, and the time-of-flight cycle was 2.4 ms. All charge states in a narrow distribution were subjected to ECD with a pulse length of 0.03 s, bias of 1.4 V, lens of 14 V, and hollow-cathode current of 1.5 A. Peak picking was performed by calculating the FTMS centroid peak on Bruker Data Analysis. Peak deconvolution was performed by auto mass peak picking on Intact Mass™ (Protein Metrics Inc., Cupertino, CA).