Autoflex max

An AutoflexMaX (Bruker Daltonik GmbH, Bremen Germany) was used. For the first measurement series, the AU samples were used as received and dropped (1 μL) on a stainless steel target. After drying, the sample holder was inserted and samples were irradiated with a Ny‐YAG Smartbeam laser working at 355 nm and 1000 Hz. Typically, 1000 laser shots from 4 different places of the spot were accumulated to a spectrum. Calibration was done using peptide standards (Bruker). FlexControl and FlexAnalysis (Bruker) were used for recording and calculating raw data.
Conditions for the second measurement series were as follows: The instrument was an AutoflexMaX (Bruker) operating at the same conditions (laser etc.) as in the first series but with a frequency of 2000 Hz. The acceleration voltage was 20 kV, a number of 2000 shots were accumulated for a spectrum, and DCTB (trans‐2‐[3‐(4‐tert‐butylphenyl)‐2‐methyl‐2‐propenyliden]‐malononitril) (Fluka) was used as matrix.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (autoflexmaX, Bruker Co., Ltd., Germany) was used to analyze the molecular weight and molecular weight distribution range corresponding to each SCSP, SCSP-I, SCSP-II, or SCSP-III sample. The sample was mixed with 2,5-dihydroxybenzoic acid (20 mg/mL) in 50% acetonitrile/water (containing 0.1% trifluoroacetic acid) at a ratio of 1:1. After 1 μL of the sample mixture was placed in one well of a 384-well plate and naturally dried at room temperature, the sample plate was placed in the ion source for measurement. The final mass spectrum was obtained by accumulating 10 single-scan signals.

To analyze the hydrolytic products of agarose or AOS produced via enzymatic reaction, MALDI–TOF/TOF MS analysis was performed in the positive ion reflectron mode using Autoflex max (Bruker Daltonics, Billerica, Massachusetts, USA), as described elsewhere (Lee et al. 2014 (link)). The enzymatic reaction mixture was spotted onto a stainless steel target plate, followed by the addition of 0.5 µL of 50% acetonitrile in which excessive 2,5-dihydroxybenzoic acid was dissolved, and air-dried. Each acquired spectrum represents the combined signal from 500 laser shots at four random locations on the spot for a total of 2000 laser shots using a 2 kHz laser. The laser attenuator offset and range were set at 18% and 40%, respectively, with the laser focus set at 36%. Mass spectra were recorded over an m/z range of 500–1500. To obtain high-resolution data, the detector sampling rate was set at a maximum of 2.50 giga samples/s, and the detector gain was set at 6.2×. flexAnalysis software (version 3.4; Bruker Daltonics) was used to process the raw MS data.