Simulated digestive peptide profiles of UHT milk and centrifugation presterilized UHT milk at different storage times
我有一个酒坑🍒: [图片] 我有一个酒坑🍒: [图片] 我有一个酒坑🍒: LC-MS/MS was used to identify the peptides released by UHT and centrifuge presterilized UHT during gastric digestion at different storage times. The first column is the peptide sequence, the second column is the UniProt id, the third and fourth column is the starting and ending position of the peptide on the corresponding sequence, and the values of the remaining columns are the abundance values of the peptide in each sample.
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The peptides released from milk during gastric digestion were identified using LC-MS/MS. For this purpose, the gastric digesta was centrifuged at 10,000 × g for 15 min and the supernatant was lyophilized. About 30 mg powder was obtained and dissolved in 1 mL 0.1% (v/v) TFA solution. After centrifugation at 12,000 ×g, the supernatant was filtered using 3 kDa molecular weight cut-off ultrafiltration centrifuge tube (Amiconfi Ultra-4, Merck Millipore, Billerica, MA, USA). Thermo Scientific Q Exactive mass spectrometer equipped with an Ultra High Performance Liquid Chromatography (UHPLC) system (Easy-nLCTM 1000, Thermo Fisher Scientific, San Jose, CA, USA) was used for the LC-MS/MS analysis. A total of 10-µL sample was loaded into a reverse-phase trap column (200 mm × 100 µm, Thermo Scientific Acclaim PepMap100, nanoViper C18, San Jose, CA, USA), which was connected to the C18-reversed analytical column (100 mm × 75 µm, 3 µm, Thermo Scientific Easy Column, San Jose, CA, USA). The gradient elution was performed with a flow rate of 300 nL/min controlled by IntelliFlow technology (Thermo Fisher Scientific, San Jose, CA, USA). The eluents used included solvent A and B; Solvent A consisted of water with 0.1% formic acid and solvent B consisted of 84% acetonitrile with 0.1% formic acid. The gradient elution program was as follows: the solvent B gradient increased linearly from 0 to 35% in 50 min, then from 35 to 100% in 5 min, and held at 100% for 5 min. The mass spectrometer was operated in the positive ion mode. A spray voltage of 2.2 kV was used with a transfer capillary temperature of 200℃. Mass spectrometry (MS) data were acquired using a data-dependent top10 method, in which, the most abundant precursor ions were dynamically selected from the survey scan (300–1800 m/z) for high-energy collisional dissociation (HCD) fragmentation. An automatic gain control (AGC) target was set at 3e6 with the maximum injection time of 10 ms. The dynamic exclusion duration was 40 s. Survey scans were acquired at a resolution of 70,000 with the m/z of 200; the resolution for HCD spectra was set to 17,500 at the m/z of 200. The precursor isolation width was 2 m/z. The normalized collision energy was 30 eV with the underfill ratio of 0.1%, which specifies the minimum percentage of target value likely to reach the maximum fill time. The instrument was run with peptide recognition mode enabled.The MS/MS spectra were analyzed using MaxQuant software version 18.104.22.168 (Max Planck Institute of Biochemistry, Martinsried, Germany) against the UniProt Bos Taurus and decoy databases. For the proteins’ identification, the parameters were set as follows: 20 ppm for peptide mass tolerance and 2 for max missed cleavage. The fixed modification included carbamidomethyl (C), while the variable modifications included oxidation (M) and acetyl (protein N-term). The cut-off value of the protein false discovery rate (FDR) for the peptide’s identification was ≤0.01.