Browsing by Subject "Mass Spectrometry"

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  • Ikonen, Jani (Helsingin yliopisto, 2020)
    At the literary review, basic concepts of proteomics and mass spectrometry were covered. Different data-collection methods (DDA and DIA) were compared with each other including exploration of the possibilities of the DIA method. Characteristics of Fourier transformation mass spectrometry were discussed in detail beginning from the production of the protein spectra in FTMS instruments including features of the Orbitrap (hybrid) mass spectrometer. Features included modes of measurements, working principle, performance characteristics, operation modes and top-down experiments including large intact protein analysis (m/z range > 6000 Da). The working principles and performance in proteomic analyses of other mass spectrometer instruments were also briefly covered. Orbitrap MS instrumentation is compared with high-performance mass spectrometers including triple quadrupole, time of flight, ion trap, and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers. Lastly, operation and coupling of the LC instrumentation to the Orbitrap mass spectrometer were also briefly discussed. The experimental part of the thesis covers development and feasibility testing of a quality control method for protein analysis studied with PierceTM Intact Protein Standard Mix by using microflow liquid chromatography-Orbitrap mass spectrometry combination. Development and testing of the method includes optimization of the method for dried sample, robustness testing with variable LC eluent concentrations, and the method performance with a heavily contaminated instrument compared with the performance of a clean MS instrument. Tested heavily contaminated instrument had more than 2000 injections of protein samples without cleaning. In the end, the developed protein analysis method was tested with nine different Q Exactive HF Orbitrap instruments to measure the instrument variation. In the studies, the average mass of analyzed proteins varied from 9111.47 to 68001.15 kDa The mass range used for identification was 500 – 2000 Da.