A protein detection technology based on the principle of enzymatic peptide hydrolysis that can verify the effect of enzymatic hydrolysis
A protein detection, protein technology, applied in the field of protein detection based on the principle of enzymatic peptide hydrolysis
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Embodiment 1
[0021] Example 1 (screening method for verifying peptides)
[0022] Take 1mg of β-lactoglobulin and dissolve in 1mL of water. Take 100 μL of the above solution, add 10 μL dithiothreitol solution (100 mmol / L) and 865 μL ammonium bicarbonate solution (500 mM) to a 2 mL plastic centrifuge tube in turn, mix well and incubate in a 70°C water bath for 30 min; add 10 μL Iodoacetamide (300mmol / L) solution, mix well, and let it stand in the dark for 30min at room temperature; add 10μL of trypsin solution, and react in a water bath at 37℃ for 10, 20, 30, 40, 50, 60, 70 , 80, 90, 120, 180, 240min; the sample was quickly added to 5 μL of formic acid solution after the reaction was completed, and filtered through a 0.22 μm filter membrane. The above samples were first screened for peptides by high-resolution mass spectrometry, and the high-performance liquid chromatography parameters were as follows:
[0023] High performance liquid chromatography: Thermo Scientific Easy-nLC1000;
[002...
Embodiment 2
[0048] Embodiment 2 (application embodiment 1 verifies the peptide to detect the content of beta lactoglobulin in the sample)
[0049] Sample pretreatment method: Weigh 10g of sample into a 100mL volumetric flask, dilute with water and make up to the mark. Take 10 μL of the above solution into a 2 mL plastic centrifuge tube, add 10 μL of isotope-specific peptide solution, 10 μL of dithiothreitol solution (100 mmol / L) and 945 μL of ammonium bicarbonate solution (500 mM) in sequence, mix well and place in a 70°C water bath Incubate for 30 min; add 10 μL of iodoacetamide (300 mmol / L) solution, mix well, and then stand in the dark for 30 min at room temperature; add 10 μL of trypsin solution, and react in a 37°C water bath for 90 min; add 5 μL of formic acid solution, pass 0.22μm membrane filtration, injection analysis.
[0050] The detection channel parameters of the above-mentioned specific peptides and verification peptides are shown in Table 2, and the liquid chromatography c...
Embodiment 3
[0061] Example 3 (screening method for verifying peptides)
[0062] For some proteins, it is difficult to completely enzymatically digest, so it is difficult to verify with the verification peptide as described in Example 1. After the specific peptide is determined in this embodiment, based on the specific peptide, the polypeptide that extends to the N-terminal or C-terminal of the specific peptide to the next enzymatic hydrolysis site is used as the verification peptide. When the signal value of the verification peptide is lower than the threshold, it can be considered as corresponding The specific peptide has been completely released from the protein.
[0063] For the specific peptide located at the N-terminus of the protein, the polypeptide extending to the C-terminus to the next enzymatic hydrolysis site can be selected as the verification peptide. For the specific peptide located at the C-terminus of the protein, the polypeptide extending to the N-terminus to the next en...
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