Protein calibration standard 1 kit

MALDI-TOF MS analysis was performed in triplicate for kimchi samples without protein extraction. First, kimchi samples were mixed and homogenized in a 1:1 ratio using α-cyano-4-hydroxycinnamic acid (CHCA) dissolved in 30% acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA). The homogenized samples with the sample matrix were dried in air and deposited on a MALDI target plate at room temperature. Mass spectra in the mass range from 2,000 to 20,000 m/z were obtained in the linear and positive-ion modes with a laser frequency of 100 Hz using an UltrafleXtreme TOF/TOF mass spectrometer (Bruker Daltonics, Germany) installed at the Daedeok Headquarters, Korea Basic Science Institute (KBSI). The extraction delay time, lens, and ion sources 1 and 2 were set at 250 ns, 7, 25, and 23.6 kV, respectively. Mass spectral data were acquired in the mzXML format using Flexcontrol and Flexanalysis software (Bruker Daltonics, Germany). Calibration for MALDI-TOF MS analysis was carried out using insulin ([M+H]⁺, 5,734.51 m/z), ubiquitin I ([M+H]⁺, 8,565.76 m/z), cytochrome C ([M+H]⁺, 12,360.97 m/z; [M+H]²⁺, 6,180.99 m/z), and myoglobin ([M+H]⁺, 16,952.30 m/z; [M+H]²⁺, 8,476.65 m/z) from the protein calibration standard I kit (Bruker Daltonics, Germany).

The acquisition of the MALDI-TOF spectra from the cheeses was made using a Microflex TOF mass spectrometer (Bruker Daltonics, Germany) equipped with a pulsed N₂ laser (337 nm, 3-ns pulse duration). The spectrometer was run with the default parameter settings: positive linear mode, 60 Hz laser frequency, ion source 1 voltage: 20 kV, ion source 2 voltage: 18 kV, and mass range of m/z 1000 to 20,000 using the FlexControl 3.0 software (Bruker Daltonics, Germany). Three independent spectra were collected manually for each cheese sample to verify the mass accuracy and reproducibility, which was always within the range of 0.5 and 1%. The external mass calibration was measured daily using the Protein Calibration Standard I kit (Bruker Daltonics, Bremen, Germany).