Two-dimensional short-gradient high-efficiency protein component separation and identification method
An identification method and protein technology, applied in the field of two-dimensional short gradient high-efficiency proteome separation and identification, can solve the problems of long analysis time, affecting the throughput and efficiency of biological sample analysis, etc.
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Embodiment 1
[0026] The Hela cells were suspended in 8M urea solution, the protein mixture was ultrasonically extracted on an ice bath, and digested with Trypsin to obtain its peptide mixture.
[0027] After digestion of 400 μg protein mixture extracted from Hela cells, one-dimensional reversed-phase chromatography was carried out. The mobile phase A used was 2% acetonitrile aqueous solution with pH 10 (adjust the pH value with ammonia water), and the mobile phase B was pH 10. 98% acetonitrile in water (adjust the pH with ammonia). The Durashell reversed-phase chromatographic separation column was purchased from Agela Company. The particle size of the reversed-phase chromatographic separation column was 5 μm, the pore diameter of the filler was 15 nm, and the reversed-phase chromatographic separation column was 4.6×250 mm. The gradient is set as the first gradient: the initial content of mobile phase B is 5%, the final content is 8%, and the gradient time is 2 minutes; the second gradient:...
Embodiment 2
[0030] C57 mice were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences. The nucleus extract obtained from the mouse liver was suspended in 8M urea solution, and its protein mixture was extracted by ultrasound on an ice bath, and digested with Trypsin to obtain its peptide mixture.
[0031] After enzymatic digestion of 400 μg protein mixture extracted from rat liver cell nuclei, one-dimensional reversed-phase chromatography was carried out. The mobile phase A used was 2% acetonitrile aqueous solution at pH 10 (adjust the pH value with ammonia water), and the mobile phase B was pH 10 98% acetonitrile in water (adjust pH with ammonia). The Durashell reversed-phase chromatographic separation column was purchased from Agela Company. The particle size of the reversed-phase chromatographic separation column was 5 μm, the pore diameter of the filler was 15 nm, and the reversed-phase chromatographic separation column was 4.6×250 mm. The gradient i...
Embodiment 3
[0034] The Hela cells were suspended in 8M urea solution, the protein mixture was ultrasonically extracted on an ice bath, and digested with Trypsin to obtain its peptide mixture.
[0035] After digestion of 400 μg protein mixture extracted from Hela cells, one-dimensional reversed-phase chromatography was carried out. The mobile phase A used was 2% acetonitrile aqueous solution with pH 10 (adjust the pH value with ammonia water), and the mobile phase B was pH 10. 98% acetonitrile in water (adjust the pH with ammonia). The Durashell reversed-phase chromatographic separation column was purchased from Agela Company. The particle size of the reversed-phase chromatographic separation column was 5 μm, the pore diameter of the filler was 15 nm, and the reversed-phase chromatographic separation column was 4.6×250 mm. The gradient is set as the first gradient: the initial content of mobile phase B is 5%, the final content is 8%, and the gradient time is 2 minutes; the second gradient:...
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