A method for outer membrane protease t to digest histones and perform mass spectrometry analysis
A technology of mass spectrometry and histone analysis, which is applied in the field of enzymolysis of histones by outer membrane protease T and mass spectrometry analysis, which can solve the problems of incomplete reaction, cumbersome operation, and indistinguishable derivative modification, etc., and achieves the advantages of simple operation and accelerated process Effect
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Embodiment 1
[0024] Example 1, the experiment of outer membrane protease T for the cleavage efficiency of lysine and arginine modified peptides
[0025] A series of standard peptides at 5 μg / μL (a total of eight, the sequence is ARTKQTA RK STGGK, where modifications on K include: no modification, monomethylation, dimethylation, trimethylation, acetylation, modifications on R include: monomethylation, symmetrical dimethylation, asymmetrical Dimethylation) was diluted 1:10 with 50mM Tris-HCl, 5mM EDTA (pH6-7) enzymatic digestion buffer to 0.5μg / μL. Take 30uL of the peptide solution, add 1-3μL of 0.1μg / μL outer membrane protease T (the ratio of peptide to enzyme is 1:50-1:100), shake and react at 25 degrees Celsius for 12 hours, heat and boil for 3- 5 minutes to stop the enzymatic reaction. After the reaction, the solution was centrifuged at high speed, and 0.8 μL of the supernatant was sampled on the MALDI target plate. After drying, an equal volume of α-cyano-4-hydroxycinnamic acid matri...
Embodiment 2
[0026] Example 2, the experiment of outer membrane protease T enzymolysis standard histone efficiency under different conditions
[0027]Prepare 50mM enzymolysis buffer with pH values of 3, 4, 5, 6, 7, 8 (3-6 is citric acid-disodium hydrogen phosphate buffer system, 7-8 is Tris-HCl), and the purified Escherichia coli Four histone mixtures were used as standard proteins, and the ratios of the four histones were basically the same. Dilute histones to 0.1 μg / μL with enzymatic digestion buffers of different pH values, heat and boil for 3 minutes to destroy the protein structure, add 1 / 100 outer membrane proteinase T after cooling, and incubate the reaction overnight at 25 degrees Celsius. After the reaction was completed, the supernatant was centrifuged at high speed, and the supernatant was desalted by a C18 column. The enzymatically hydrolyzed peptide was redissolved in 0.1% FA, and then analyzed by LTQ-OrbitrapXL mass spectrometry. The temperature conditions were set as: 15°...
Embodiment 3
[0028] Example 3, an experiment based on the outer membrane protease T one-step enzymatic hydrolysis of histones and the effect of mass spectrometry analysis
[0029] Take the 293T cells collected in a 10cm dish, wash 2-3 times with 500μL-1mL PBS, add 500μL lysate (150mM NaCl, 20mM Tris-HCl pH7-8, 0.1% Triton-100 and protease inhibitors) and mix thoroughly The cells were broken, and the precipitate was collected by centrifugation at 10,000 g; then 800 μL of 0.4M HCl was added and incubated overnight at four degrees. The next day, the supernatant was obtained by high-speed centrifugation at 12,000g and transferred to a 3KDa ultrafiltration tube. 2 After washing twice, replace with 50 μL 50mM Tris-HCl pH6-7, 5mM EDTA enzymatic digestion buffer; after quantification by BCA quantification kit, dilute the obtained histones with enzymatic digestion buffer to 1 μg / μL, according to the ratio of 1:25 Add outer membrane proteinase T and react at 37 degrees Celsius for 14 hours. Afterw...
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