Developmental changes in proteins of casein micelles in goat milk using data-independent acquisition-based proteomics methods during the lactation cycle
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Developmental changes in proteins of casein micelles in goat milk using data-independent acquisition-based proteomics methods during the lactation cycle
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The dried tryptic peptides were resuspended in 0.1% FA and subjected to EASY-nLC 1000 coupled with Orbitrap Fusion Lumos (Thermo Fisher Scientific, Milford, MA, USA). The column was equilibrated with buffer A (0.1% FA). The peptides were loaded onto a C18 trap column (100 μm × 20 mm, 5 μm; Thermo Fisher Scientific) using an autosampler and separated using a C18 analytical column (75 μm × 150 mm, 3 μm; Thermo Fisher Scientific) at a flow rate of 300 nL/min. The separation gradient was as follows: buffer B (80% acetonitrile and 0.1% FA) from 4% to 10% within 5 min, from 10% to 30% within 58 min, from 30% to 40% within 9 min, from 40% to 100% within 8 min, and finally, holding at 100% for 10 min. For DDA analysis, mass spectrometry was performed in positive ion mode with a parent ion scanning range of 300–1,800 mass/charge (m/z) and automatically switched between MS and MS/MS acquisition. The parameters of mass spectrometry were set as follows: (1) MS: resolution, 60,000; AGC target, 400,000; maximum injection time, 50 ms; and exclusion duration, 40 s. The top 20 most abundant precursor ions with a charge of ≥2 from the MS scan were selected and fragmented by high-energy collisional dissociation with normalized collision energies of 27 eV. (2) HCD-MS/MS: resolution, 15,000; AGC target, 50,000; maximum injection time, 50 ms. For DIA analysis, mass spectrometry was performed in positive ion mode with a parent ion scanning range of 395–1205 m/z. The mass spectrometry parameters were set as follows: (1) MS: resolution, 60,000; AGC target, 2e6; and maximum injection time, 100 ms. (2) HCD-MS/MS: resolution, 15,000; AGC target, 1e6; collision energy, 30 eV. (3) A DIA using an isolation width of 26 Da (containing 1 Da for the window overlap) and 32 overlapping windows was constructed covering the precursor mass range of 400–1200 Da for DIA acquisition according to a previous study (Jin et al., 2021).