The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.
Our protocol uncovers epigenetic markers for sorghum plant development and drought resistance. Such molecular understanding will help develop better solutions to adapt crops for extreme climates in the future. This protocol generates high purity histones from sorghum leaf tissue suitable for un-targeted profiling of post-translational modifications by mass spectrometry.
Demonstrating the procedure will be Shadan Abdali and Tanya Winkler, post-bachelor research associates from EMSL. To begin, grind a few sorghum leaves with liquid nitrogen and store them in a 50 milliliter centrifuge tube at minus 80 degrees Celsius. Use approximately four grams of the leaf powder for histone analysis of each sample.
Prepare extraction buffers 1, 2A, and 2B as described in the text manuscript and add a protease inhibitor tablet to extraction buffer one to make a final concentration of 0.2X. Then add 20 milliliters of the prepared extraction buffer one to the ground leaf powder and gently mix or vortex for 10 minutes. Using mesh 100, rinse the filtered material twice with two milliliters of extraction buffer one each time.
Next, centrifuge the filtrate at 3, 000 times G for 10 minutes at four degrees Celsius in a swinging bucket rotor to pellet the cell debris and large subcellular organelles. Simultaneously, prepare extraction buffer 2A by adding protease inhibitor to a final concentration of 0.4X. Discard the supernatant without disturbing the pellet.
Then resuspend the pellet in five milliliters of the prepared extraction buffer 2A. Incubate it on ice for 10 minutes with gentle mixing. Centrifuge the solution at 2, 100 times G for 15 minutes at four degrees Celsius in a swinging bucket rotor to pellet the debris and nuclei.
Decant the supernatant carefully without disturbing the pellet. Prepare extraction buffer 2B by adding the protease inhibitors to a final concentration of 1X. Add five milliliters of this prepared buffer to the pellet obtained after the centrifugation.
Centrifuge at 2, 100 times G for 15 minutes at four degrees Celsius in the swinging bucket rotor to pellet debris and nuclei. Simultaneously, prepare nuclei lysis buffer by adding a protease inhibitor tablet. Carefully decant the supernatant without disturbing the pellet and resuspend the pellet in 250 microliters of the lysis buffer.
Vortex the solution for 15 seconds at maximum speed to homogenize it and resuspend the material. Then sonicate it for five minutes at four degrees Celsius and store it at minus 80 degrees Celsius. Thaw the frozen nuclei sample and add 750 microliters of 5%guanidine buffer.
Then sonicate it for 15 minutes at four degrees Celsius. Transfer the sample to a two milliliter tube and spin it at 10, 000 times G for 10 minutes at four degrees Celsius. Simultaneously, prepare an ion exchange chromatography column by rinsing it with two milliliters of acetonitrile and four milliliters of water to minimize contamination on the surface.
Load approximately 200 to 300 microliters of weak cation exchange resin onto the chromatography column and let the resin settle. Wash the resin four times with guanidine buffer and place the tube and column on ice. Put the column on a two milliliter collection tube and load the supernatant from the prepared sample slowly onto the resin bed without disrupting the resin.
As the solution is flowing through, load the eluent back to the top of the column six to eight times to allow maximum binding to the resin. Then load two milliliters of 5%guanidine buffer to wash the non-histone proteins off the column. Elute the histone proteins with one milliliter of 5%guanidine buffer and collect the eluent.
Clean a three kilodalton cutoff spin filter with 500 microliters of wash solvent, then desalt the remaining eluent using the pre-cleaned spin filter. Major histone proteins were observed in specific regions of the LC-MS feature map indicating the success of the experiment. This representative map shows intact full-length histones in dashed boxes.
The LC-MS feature map of H2B and 16 kilodalton H2A proteoforms shows that both have multiple homologs with similar sequences as noted by the unique product session numbers. Another group of H2A histones without the extended tails can be seen. The N-terminal acetylation, additional lysine acetylations, and methionine oxidations in H4 histone proteins can be observed by examining the mass differences in the LC-MS feature map shown here.
There were two protein sequences identified for H3 histone proteins, H3.3 and H3.2. Electron transfer dissociation fragmentation was used to generate the fragmentation spectrum of the identified H3.2 proteoform. The precursor ions and the previous and next Ms1 spectra are shown here with their matched isotope peaks highlighted in purple.
Post-translational modifications can be localized using the sequence coverage map. By comparing the relative abundance of the proteoforms, changes of truncated histone proteoforms specific to sample conditions were discovered. C-terminal truncation of H4 was observed only in weeks three and nine for some of the samples.
For H3.2, N-terminal truncated proteoforms were generally more abundant in week 10. In contrast, C-terminal truncated H3.2 were seen in earlier time points. The H4 C-terminal truncated proteoforms were significantly more abundant in BTx642 than in RTx430.
When attempting this protocol, pay close attention to the colors of the supernatant and the pellet. The color change help identify potential problems in the grinding or lysis of the chloroplast. We hope that this robust protocol for histone isolation from sorghum leaves will enable epigenetic research for other similar plants.
