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Supporting Information Supporting Information ORIGAMI: A Software Suite for Activated Ion Mobility Mass Spectrometry Applied To Multimeric Protein Assemblies Lukasz G. Migas, Aidan France, Bruno Bellina and Perdita E. Barran* Michael Barber Centre for Collaborative Mass Spectrometry, School of Chemistry and Manchester Institute of Biotechnology The University of Manchester, 131 Princess Street, Manchester, M1 7DN, United Kingdom Correspondence and request for materials should be addressed to *Professor Perdita Barran (email: [email protected]) Table S1. Experimental parameters from TWIMS experiments Source conditions and gas flows Capillary voltage (kV) 0.7 – 1.2 Source temperature (°C) 40.0 Sample cone (V) 10.0 – 20.0 Extractor cone (V) 1.0 Source gas flow (mL/min) 30.0 Trap gas flow (mL/min) 2.0 Helium cell gas flow (mL/min) 200 IMS gas flow (mL/min) 75.0 DC voltages Trap cell energy (V) 4.0 – 200.0 Trap DC entrance 3.0 Trap DC bias 44.0 Trap DC -2.2 Trap DC exit 4.0 Trap height (V) 15 IMS DC entrance 27.0 Helium cell DC 30.0 Helium cell exit -5.0 IMS bias 2.0 IMS DC exit 0.0 Transfer cell energy manual control OFF Transfer cell energy (V) 0.0 – 6.0 Transfer DC entrance 0.5 Transfer DC exit 10.0 Table S2. Typical ORIGAMIMS parameters and their description. Variables Typical values Comments Scans per 3 Number of scans spent on each CE voltage. Voltage Increase if your signal intensity is initially low Scan time (s) 5 Typically set to MassLynx maximum (5 s) as this reduces data size and analysis time Start Voltage (V) 4 First voltage in the CIU acquisition. Adjust depending on sample and needs End Voltage (V) 200 Final voltage in the CIU acquisition. Adjust depending on sample and needs Step Size (V) 0.1+ Voltage increment between Start and End Voltage. Small values capture more finite detail but elongate run time Ion Polarity Positive/Negative Select ion polarity mode Activation Zone Sampling Cone/Trap Select the pre-quad Sampling Cone or post- quad Trap Collision Cell Ramp Type Linear/Exponential/Fitted/User- Collision voltage ramp type responsible for defined controlling the number of scans per voltage Exp % 0-100 % Relative time at which the exponential method starts increasing the value of SPV. Exp increment 0-0.035 Increment value determine the rate of increase in the exponential method Fitted slope 20-80 Value defining the rate at which SPV increases according to the modified Boltzmann equation User-specified - A list of SPV values to be used for each input collision voltage Figure S1. ORIGAMIMS graphical user interface. The software is an interface between WREnS and the EPC on Waters IM-MS instrument and sits alongside MassLynx. The user has the choice to select the ion polarity, activation type (ion source or trap collision cell) and collision voltage ramp type (linear, exponential, fitter or user-specified). A number of experimental parameters include: scans per voltage (SPV), scan time, start, end and step voltages and method specific parameters. Specific modes are shown in (a-c); a) linear acquisition mode where the number of scans per voltage remains constant; b) exponential acquisition mode where the SPV increases as the collision voltage increase based on the exponential curve. User parameters include Exp % which determines the relative starting point of the increase and Exp increment which controls the slope of the increase; c) fitted acquisition mode where SPV increases based on a modified Boltzman equation with user controlling the relative starting point using the fitted slope parameter. ANALYSE Figure S2. ORIGAMI graphical user interface. The typical view of the analysis software in this case focusing on the parent and fragment ions of ConA 20+ ANALYSE [4M+20H] . ORIGAMI can operate on a single or a list of MassLynx files, permitting performing identical analysis on separate files essentially reducing the uncertainty introduced during standard protocol. ANALYSE ANALYSE Figure S3. ORIGAMI graphical user interface utilsing the overlay method to distinguish between two text files. ORIGAMI can operate on a single or a list of text files and these can be compared directly with MassLynx .raw files. Figure S4. Inclusion of ORIGAMIMS in the day-to-day collision induced unfolding experiments is relatively simple. The user can control all of mass spectrometry conditions in MassLynx, whilst the CIU parameters (cone voltage or trap collision voltage) are controlled via ORIGAMIMS. The CIU protocol typically results in a ion chromatogram shown above, where as the collision voltage increases, the presence of the precursor ion decreases and appearance of fragment ions is more prevalent. The example shown above is the Alcohol dehydrogenase 24+ with 3 SPV, 4-180 V ramp range with a step size of 2 V. .
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