Ultraflex 3 instrument

MTs were purified by using FPLC according to our previous study [44 (link)]. After isolation, MTs were analyzed using MALDI-TOF MS (Ultraflex III instrument, Bruker Daltonik, Leipzig, Germany) equipped with a laser, operating at wavelength of 355 nm with an accelerating voltage of 25 kV, a maximum energy of 43.2 µJ, and a repetition rate of 2000 Hz. The matrixes used for analyses were α-cyano-4-hydroxycinnamic acid (HCCA) and 2,5-dihydroxybenzoic acid (DHB) (Bruker Daltonik, Leipzig, Germany) prepared in acetonitrile solution (30% w/w) with the addition of trifluoroacetic acid (0.1% w/w). Matrix and substance solutions for analysis were mixed in ratio of 1:1 (v/v). Obtained homogeneous solution (1 µL) was dried under atmospheric pressure and ambient temperature (25 °C). MS spectra were typically acquired by averaging 20 sub-spectra from a total of 500 shots of the laser (Smartbeam 2. Version: 1_0_38.5, Bruker Daltonik, Leipzig, Germany).

The identities of the compounds were confirmed by the following methods. Both ¹H-NMR (600 MHz) and ¹³C-NMR (100 MHz) spectra were recorded using a JEOL JNM-ECA 600 (JEOL Ltd., Tokyo, Japan) (and JNM-ECA 920 (JEOL Ltd., Tokyo, Japan)) spectrometer, respectively. The spectra were measured in CDCl₃ solvents at room temperature, DMSO-d₆ and D₂O solvents at 40 °C and 4,4′-dimethyl-4-silapentane-1-sulfonate (DSS) as an internal standard. MALDI TOF mass spectra were measured by a Bruker Ultraflex III instrument (Bremen, Germany) with a 337 nm nitrogen laser. The dendrimer sample solutions (1 μL, 1 mg/100 μL of MeOH:THF = 1:1) and the matrix solution (1 μL, 2,5-dihydroxybenzoic acid of 2 mg/100 μL MeOH:THF = 1:1) were applied to the 24 × 16 well ground steel MALDI probe (Bruker Daltonics K.K., Yokohama, Japan). The dendrimers sample solution (1 μL, 1 mg/100 μL of 0.1% (v/v) TFA in ACN:H₂O = 2:1) and the matrix solution(1 μL, 2,5-dihydroxybenzoic acid of 2 mg/100 μL of 0.1% (v/v)TFA in ACN:H₂O = 2:1) were applied to the 24 × 16 well ground steel MALDI probe. The sample was dried by air evaporation and then the nitrogen laser was irradiated to each sample to obtain the corresponding mass spectrum.