2.3. Mass Spectrometry

MALDI-TOF (matrix assisted laser desorption/ionization-time of flight) mass spectra were recorded in linear mode by means of a Bruker Ultraflex III MALDI-TOF/TOF instrument equipped with a Nd:YAG laser at a wavelength of 355 nm with <500 ps pulse and 200 Hz firing rate. The accelerating voltage was 22 kV. External calibration was performed using a Bruker calibration mixture consisting of polypeptides with different molar mass values. All measurements were performed in positive ion mode. Approximately 2000 laser shots were accumulated for each mass spectrum, and 2,5-dihydroxybenzoic acid was used as the matrix in the analysis of the samples.

Mass spectra in reflectron mode were recorded by means of a 4800 Proteomic Analyzer (Applied Biosystems, Foster City, CA, USA) MALDI-TOF/TOF instrument equipped with a Nd:YAG laser at a wavelength of 355 nm with <500 ps pulse and 200 Hz firing rate. The accelerating voltage was 20 kV. External calibration was performed using an Applied Biosystems calibration mixture consisting of polypeptides with different molar mass values. The irradiance was maintained slightly above the threshold, to obtain a mass resolution of about 7000–10,000 fwhm; isotopic resolution was observed throughout the entire mass range detected (from m/z 500 up to m/z 5000). Mass accuracy was about 40 ppm. All measurements were performed in positive ion mode; approximately 1500 laser shots were accumulated for each mass spectrum in reflectron mode, and 250 laser shots in linear one too. For the analysis of all the samples several matrices such as DHB, DCTB and dithranol, were used with and without salts as cationizing agent (i.e., CF₃COOLi; CF₃COONa). All matrices were solubilized in THF at a concentration of 0.1 M, poly-6-MOEG-9-T-BF3k samples were solubilized in the appropriate solvent (i.e., CHCl₃ or DMF) with a concentration of 2 mg/mL. Samples for MALDI analysis were prepared by the dried-droplet method, in which a mixture of matrix and sample was deposited onto the target plate and dried at room temperature under inert atmosphere (N₂ flow), and also by the multilayer method, in which firstly a layer of matrix solution (0.3 µL) onto the target plate then after 10 min a layer of polymer solution (0.2 µL) was deposited and finally after 1 h a final layer of matrix solution (0.3 µL) was added. The best spectra were recorded using DCTB as matrix with or without CF₃COOLi as doping agent.