Pregunta general

6.6.1 Data acquisition

Data were acquired by means of a Synapt HDMS System in ESI positive mode with a capillary voltage of 1.2 kV from 1000-4500 m/z. The time-of-flight (TOF) mass analyser was calibrated using 2 mg/ml cesium iodide in 50% aqueous propan-2-ol. Instrument acquisition parameters were adjusted to provide the optimal ion mobility separation. The cone voltage was 60 V and the collision energy in the trap region was 10 eV. Source temperature and gas flow were 110°C and 35 ml/min respectively. Nitrogen was used as the gas in the ion mobility cell and the indicated pressure within the cell was 0.68 mbar, equivalent to a flow rate of 38 ml/min. The backing pressure was increased in increments from 2 to 8 mbar to identify the ideal pressure conditions for transmission of the relevant ionic species. The travelling wave velocity and wave height were altered in increments from 100-600 m/s and 8- 20V respectively, and the conditions that provided the optimal mobility separation were used for all following experiments.

The synchronisation of gated release of ions into the ion mobility separator with TOF acquisition allows arrival time distributions of ions to be obtained. Ions are released from the trapping cell to the mobility cell in a pulse every 100 μs, and 200 orthogonal acceleration pushes of the TOF analyser are recorded to form one ion

mobility experiment. The overall mobility recording time is 200 x tp, where tp is the

pusher period (Pringle et al., 2007). The pusher period depends on the mass acquisition range; for these experiments, a pusher period of 120 µs was used giving a mobility recording time of 24 ms. Equine myoglobin at a concentration of 10µM in 50% aqueous acetonitrile containing 0.2% formic acid was used to provide data which were used to create a calibration curve for cross-sectional measurements. Data obtained for each hemoglobin tetramer over the m/z range 3000-4500 were deconvoluted on to a true mass scale using the MaxEnt software to provide an estimate of molecular mass. Experiments were carried out in triplicate.

6.6.2 Calibration, modelling and cross-section estimation

The equine myoglobin data was used to create a calibration curve for each set of experiments. Absolute cross-sections for equine myoglobin were obtained from drift-time ion mobility mass spectrometry (DTIMS) studies (Prof. Michael T. Bowers, personal communication). The calibration was performed using a procedure developed in-house based on previously published work (Ruotolo et al., 2005; Scrivens et al., 2006; Wildgoose et al., 2006). In brief, normalised cross-sections (corrected for charge and reduced mass) were plotted against corrected arrival times (corrected to exclude time spent outside the ion mobility cell) to create a calibration with a power series fit. The calibration allows for estimation of the cross-section of a molecule of interest provided that the mobilities (corrected arrival times) for that molecule lie within the mobilities observed for the calibrant, irrespective of the size range of cross-sections for the calibrant (Shvartsburg and Smith, 2008; Thalassinos

et al., 2008). The calibration was used to estimate rotationally averaged collision cross-sections of hemoglobin monomer, dimer and tetramer for the different charge states observed based on their arrival time distributions, provided that their corrected arrival times fell along the calibration curve.

To compare the experimental cross-sections for the normal and sickle hemoglobin tetramers with accepted values, cross-sections were calculated using MOBCAL, a program to calculate mobilities (Meslehet al., 1996; Shvartsburg and Jarrold, 1996) from published X-ray structures held at the RCSB Protein Data Bank (Berman et al., 2000). MOBCAL facilitates the use of three approximations to calculate cross

sections. The projection approximation (PA) typically results in an underestimation of the cross-section of a large ion. It calculates the cross-section by averaging the projections produced by every orientation of a molecule and so does not take into account interactions with the buffer gas. The trajectory method (TM) takes into account all interactions but is computationally intense. The exact hard sphere scattering model (EHSS) carries out trajectory calculations whilst ignoring long- range interactions but nevertheless gives values within a few percent of the TM approximations (Jarrold, 1999; Scarff et al., 2008). For this work, cross-sections were calculated using the PA and EHSS methods to reduce computational time.

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Chapter 7