Ammonia solution 25

Methyl methacrylate, glycidyl methacrylate, oligo(ethylene glycol) methyl ether methacrylate (OEGMA), 4-cyano-4(phenylcarbonothioylthio) pentanoic acid (CPADB) (>97%), azobisisobutyronitrile (AIBN), propargyl alcohol, sodium azide, N,N’-dicyclohexylcarbodiimide, 4-dimethylaminopyridine, copper(I)bromide, N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA), ammonium chloride, ammonia solution (25%), toluene, methanol, methylene chloride, diethyl ether, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, choloroform, and neutral alumina were purchased from Sigma-Aldrich and were used as received. NMR solvents CDCl₃ and DMSO-d₆ were purchased from Eurisotop, Saint Aubin, France.

Nusinersen is the sodium salt of an 18-mer 2’-O-methyl-phosphorothioate oligoribonucleotide dissolved in sodium dihydrogen phosphate dihydrate, disodium phosphate, sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium hydroxide, hydrochloric acid, and water (Spinraza™). The full sequence of the ASO is 3’-mU-mC-A-mC-mU-mU-mU-mC-A-mU-A-A-mU-G-mC-mU-G-G-5′, while the molecular mass equals 7110 g/mol.
Mobile phases were prepared using high purity solvents such as methanol, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), as well as N,N-dimethylbutylamine (DMBA), N,N-diisopropylethylamine (DIPEA), dipropylamine (DPA) and LC-MS water (Merck KGaA, Darmstadt, Germany). The mixture of phenol/chloroform/isoamyl alcohol (25:24:1) (VWR International, Gdańsk, Poland), acetonitrile, chloroform, 10 mM ammonium acetate (pH 4.5 and 9.0), acetic acid, ammonia solution 25% (Sigma-Aldrich, Gillingham, Dorset, UK) was also used during sample preparation step.

The wild-type (WT) 307–319
segment of the TDP₄₃ protein (TDP-43_307–319) with sequence H₂N-³⁰⁷MGGGMNFGAFSIN³¹⁹-C=ONH₂ was purchased from Biomatik and used without
further purification (>98%). Ammonium acetate (AA) solution 5 M
in
H₂O and ammonia solution 25% were purchased from Sigma-Aldrich.
Initially, 1.5 mg of the WT peptide was dissolved in 0.8 mL of 1,1,1,3,3,3-hexafluoro-2-propanol
(HFIP, LC–MS grade, > 99.8%, Sigma-Aldrich) and sonicated
for
5 min to ensure total dissolution of the sample. This solution was
then divided in 50 μL aliquots that were partially covered and
left to dry at room temperature for approximately 15 h. Once the HFIP
was fully evaporated, the aliquots were stored at −20 °C.
A stock solution of 100 μM was prepared using 10 mM ammonium
acetate (AA) buffer with pH 7.4 and 2% of HFIP. The pH of the buffer
was adjusted to pH 7.4 using a 0.5% ammonia:water solution of pH 11.4.
The stock solution was further diluted using the same AA buffer to
a final concentration of 20 μM to be used in the TIMS-qToF for
analysis. The WT samples were stored at room temperature and were
analyzed after 1–7 days of their preparation.