TITLE: Histone extraction for mass spectrometry-based analysis of post-translational modifications in the fungal genus Aspergillus
AUTHORS: Xin Zhang, Roberta Noberini, Tiziana Bonaldi, Michael    F. Seidl, Jérȏme    Collemare

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Mass spectrometry is a powerful technique to uncover histone post-translational modifications in an unbiased and quantitative manner (Noberini,</span><span style = "font-style:italic;">et al</span><span>.,The FEBS Journal, 2021). Yet, such analyses require good quality protein samples, a challenge when working with fungi. Here, we present a protocol based on the work by Noberini and colleagues (Noberini, </span><span style = "font-style:italic;">et al</span><span>., Methods, 2020) to extract histone proteins from filamentous fungi for applications like mass spectrometry-based analyses of post-translational modifications. Several modifications were made: 1) additional cell disruption and homogenization steps using freeze-drying and TissueLyser II; 2) increased Triton X-100 concentration in the lysis buffer; and 3) prolonged incubation time in lysis buffer. This protocol is reproducible, and the proteins extracted from three </span><span style = "font-style:italic;">Aspergillus</span><span> species (</span><span style = "font-style:italic;">A. niger</span><span>, </span><span style = "font-style:italic;">A. fumigatus,</span><span> and </span><span style = "font-style:italic;">A. nidulans</span><span>) were of good quality for mass spectrometry.</span></div></div>

[STEPS]
?. [Sample collection and disruption]
Harvest fungal spores from 4- or 5 -day old sporulating plates with a spreader in 12.5 mL ice-cold ACES buffer
?. [Sample collection and disruption]
Discard supernatant and resuspend spores in 25 mL ice-cold ACES buffer.
?. [Sample collection and disruption]
Resuspend spores in 10 mL ice-cold ACES buffer, and count spore concentration with an hemacytometer or spore counter. Ideally, the spore concentration is around 1  × 107 spores/ mL.
?. [Sample collection and disruption]
Inoculate 50 mL of liquid medium with 1 ~ 10 × 107 spores in a 250 mL flask, and incubate at 30 ℃, 200 rpm, for 16 hours.
?. [Sample collection and disruption]
Collect germinated spores by centrifugation at 4,000 xg for 10 minutes and discard supernatant.
?. [Sample collection and disruption]
Resuspend germinated spore pellet in 25 mL 0.8 M NaCl.
?. [Sample collection and disruption]
Freeze germinated spores in liquid nitrogen.
?. [Sample collection and disruption]
Grind freeze-dried samples with a mortar and pestle, and then with TissueLyser II using two ⌀5 mm metal beads at 30 Hz for 30 seconds, which is repeated twice.
?. [Histone protein extraction]
Resuspend 100 mg ground sample in 1 mL lysis buffer with high Triton X-100 concentration (1×PBS, 0.5 mM PMSF, 5 µM Leupeptin, 5 µM Aprotinin, 5mM Na-butyrate, and 10% Triton X-100).
?. [Histone protein extraction]
Add 10 ml lysis buffer with low Triton X-100 concentration (1×PBS, 0.5 mM PMSF, 5 µM Leupeptin, 5 µM Aprotinin, 5mM Na-butyrate, and 1% Triton X-100), mix by vortexing, and incubate on the orbital shaker at 4 ℃ for another 30 minutes.
?. [Histone protein extraction]
Centrifuge at 3,250 xg for 30 minutes at 4 ℃, and discard supernatant.
?. [Histone protein extraction]
Add 9 mL ice-cold PBS buffer and mix by vortexing.
?. [Histone protein extraction]
Resuspend pellet in 2 mL 0.4 N H2SO4 and incubate at 4 ℃ for 4 hours on an orbital shaker.
?. [Histone protein extraction]
Centrifuge at 16,000 xg for 10 minutes at 4 ℃. The supernatant contains soluble core histone proteins.
?. [Histone protein extraction]
Add 4 volumes of acetone to the sample, and keep at -20 ℃ overnight to precipitate histone proteins.
?. [Histone protein extraction]
Centrifuge at 6,000 xg for 25 minutes at 4 ℃ to pellet proteins, then dissolve in 100 µL water.
?. [Protein quantity and quality measurement]
Measure protein concentration with the Bradford Protein Assay (He, F., Bio-protocol, 2011).
?. [Histone protein extraction]
Sonicate for 30 seconds; this process is repeated 10 times with 30 seconds break in between.
?. [Protein quantity and quality measurement]
Check protein quality via 16% SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel (Simpson, R. J., Cold Spring Harbor Protocols, 2006).
?. [Sample collection and disruption]
Centrifuge the spore suspension at 2,500 xg at 4 ℃ for 5 minutes.
?. [Sample collection and disruption]
Centrifuge the spore suspension at 2,500 xg at 4 ℃ for 5 minutes, and discard supernatant.
?. [Sample collection and disruption]
Freeze dry overnight.
?. [Histone protein extraction]
Incubate on an orbital shaker for 30 minutes at 4 ℃.
?. [Sample collection and disruption]
Centrifuge at 4,000 xg for 10 minutes and discard supernatant to get rid of liquid medium.
?. [Histone protein extraction]
Centrifuge at 3,250 xg for 20 minutes at 4 ℃, and discard supernatant.
?. [Histone protein extraction]
Resuspend in 1 mL ice-cold PBS buffer.