Unaided trifluoroacetic acid pretreatment solubilizes polyglutamine peptides and retains their biophysical properties of aggregation

Insights into the molecular mechanism behind solubilization of amyloidogenic polyglutamine‐containing peptides

“…The pure fraction of the peptide was collected in one glass vial (Borosil, 15 mL) based on its retention time and previously characterized mass spectrometry data (Supporting Information Figure S1b and Table S1). They were then freeze‐dried by using a lyophilizer and stored at −80 °C for future use …”

“…After incubation, the volatile solvents were removed using nitrogen gas, and their removal was ensured by further subjecting them to vacuum drying for 1 hour. The peptide stock solutions were generated by dissolving the dry peptide film in water‐TFA (pH 3.0) . Any insoluble material or aggregates were removed by subjecting the resultant peptide solution to ultracentrifugation at 350 300 g for 4 hours.…”

“…The supernatant was carefully taken without disturbing the bottom one third of the solution. The extent of disaggregation under different solvent pretreatment conditions was determined by measuring the percent monomer present in the supernatant based on the RP‐HPLC analysis …”

“…A final spectrum is an average of 120 scans at 4 cm −1 resolution recorded under constant purging of nitrogen gas at room temperature. The amide I region of primary spectra was selected and subjected to second derivative analysis after 9‐point smoothing by using Opus 7.2 software provided along with the instrument …”

“…One such exception was the polyGln peptide with flanking sequences that represent the N‐terminal exon 1 encoded region in a full‐length huntingtin protein. The optimal yield for this peptide was found to be when it was disaggregated at 0.1 mg/mL concentration . However, both the studies that use TFA and TFA/HFIP have not explained the molecular mechanism behind disaggregation and effect of these solvents on polyGln peptides and their aggregation.…”