Ultraflextreme maldi tof tof mass spectrometry

The molecular mass-to-volume relationship of TROSPA_NΔ44 was initially assessed by SEC (as described above), using BSA, lysozyme and TEV protease as standards. SEC was performed under both native [in a buffer composed 25 mM Tris-HCl, pH 8, 200 mM NaCl and 1 mM Tris (2-carboxyethyl) phosphine] and denaturing conditions [in a buffer composed 25 mM Tris-HCl, pH 8, 200 mM NaCl, 1 mM Tris (2-carboxyethyl) phosphine and 8 M urea] at 20 °C. The fractionated proteins were identified by SDS-PAGE. The M_m of TROSPA_NΔ44 was also determined using Ultraflextreme MALDI Tof/Tof mass spectrometry (Bruker Daltonics). The polydispersity and R_h of the protein were determined by dynamic light scattering (DLS) using a Zetasizer μV (Malvern). The measurements were performed immediately after the concentration and filtration steps using a 4-μl sample in a 1-cm path-length quartz cuvette (Hellma QS 105.231). DLS measurements were also employed to estimate the thermostability of the protein samples and the changes in the R_h at different temperatures. The heating rate was 1 °C min⁻¹, data were collected every 2 °C, and the measurements began after a 5-min equilibration at each step. Thirteen measurements were taken for each temperature (ranging from 4 °C to 80 °C).

MALDI TOF MS analysis was performed by a Bruker ultrafle Xtreme MALDI TOF/TOF mass spectrometry (Bruker Daltonics, France). α-cyano-4- hydroxycinnamic acid (HCCA) was used as the matrix and was dissolved into ACN. The extracted proteins were dissolved into water and 1 µl of protein solution was mixed with 1 µl of matrix. The mixture was dripped on a reusable polished steel target and left to dry. The ion mode was positive ion mode with delay: 150 ns; ion source 1 voltage: 20 kV; ion source 2 voltage: 18 kV; lens voltage: 6 kV. All spectra were shown baseline-subtracted, smoothed, and auto-scaled in the Y-direction, covering a range of 300–3,000 Da.