Asp n

ΔNSALL2-Flag was immunoprecipitated from Flp-In™ T-REx™ U2OS cells transfected with pCMV(NH)/ΔNSALL2-Flag, treated with DMSO or 30 μM Silmitasertib for 6 h. For mass spectrometry analyses of in vitro phosphorylated SALL2, cold ATP was added to the 5X reaction buffer for in vitro phosphorylation assays described above.
Samples were subjected to polyacrylamide gel electrophoresis followed by gel staining and extraction with the MASSPrep Automated Digestor (Waters/Micromass) from the London Regional Proteomics Center at the University of Western Ontario, Canada. After removing the dye, immunoprecipitated ΔNSALL2-Flag was digested with Trypsin in 50 mM NH₄HCO₃ solution, followed by digestion with Asp-N (Sigma-Aldrich). Peptides obtained from digested samples were extracted from the gel and sent to London Regional Proteomics Center for Mass Spectrometry analyses. The samples were subjected to tandem mass spectrometry in the Q-tof Ultima Global equipment (Micromass), with electrospray ionization (ESI) and fragmentation by activated/collision-induced dissociation (CAD/CID). The analyses were performed in PEAKS Studio software [93 (link)] in the UniProt database for Homo sapiens, with a 1% FDR (false discovery rate).

Purified protein (2 µg) was diluted in 25 mM NH₄HCO₃ and incubated with 0.05% (final concentration) Rapigest SF Sufactant (Waters, Manchester, UK) at 80°C for 10 min. To reduce disulfide bonds, DTT in 25 mM NH₄HCO₃ was added to a final concentration of 10 mM and incubated at 60°C for 10 min. The sample was then carbamidomethylated with iodoacetamide (3 mM final concentration) in 25 mM NH₄HCO₃ and incubated at room temperature for 30 min in the dark. The protein was then digested at 37°C overnight by addition of sequencing grade trypsin or endopeptidase Glu-C (Roche, Lewes, UK) in 25 mM NH₄HCO₃ to a protein : enzyme ratio of 50 : 1. Digestion with endopeptidase Lys-C (Roche, Lewes, UK) followed a similar protocol using 25 mM Tris HCl, 1 mM EDTA pH 8.5 as the digestion buffer. Digestion with Asp-N (Sigma-Aldrich, UK) was performed in 50 mM AmBic pH 8.0 with a protein : enzyme ratio of 20 : 1.

A total of 20 μg of purified protein was reduced, S-alkylated, and digested with trypsin (Promega). If required, samples were additionally digested with the endoprotease Asp-N (Sigma-Aldrich). Glycopeptides were then analyzed by capillary reversed-phase chromatography and electron-spray MS using a Bruker Maxis 4G Q-TOF instrument as described previously (18 (link)). Site-specific glycosylation occupancy was calculated using the ratio of deamidated to unmodified peptide determined upon N-glycan release with peptide:N-glycosidase A (Europa Bioproducts).

The synthesized linear GeXXVIIA was alkylated with 10 mM iodoacetamide (IAA) in 100 mM Tris-HCl, pH 8.7, and 2 mM EDTA at 22 °C in the dark for 1 h. The alkylated linear peptide GeXXVIIA-L was purified on a ZORBAX C18 HPLC analytical column (4.6 × 250 mm, Agilent). The digestion of alkylated GeXXVIIA-L was carried out in 100 mM Tris-HCl, pH 8.5, using 3 μg/mL Asp-N (Sigma, St Louis, MO, USA) at 37 °C for 18 h. The digested products were separated on a ZORBAX C18 HPLC analytical column (4.6 × 250 mm, Agilent).

A total of 20 µg of monomeric purified protein was reduced, S-alkylated, and digested with trypsin (Promega). If required, samples were additionally digested with the endoproteinase Asp-N (Sigma-Aldrich). Glycopeptides were then analysed by capillary reversed-phase chromatography and electron-spray MS using a Bruker Maxis 4G Q-TOF instrument as described previously (Grünwald-Gruber et al., 2017 (link)). Site-specific glycosylation occupancy was calculated using the ratio of deamidated to unmodified peptide determined upon N-glycan release with peptide:N-glycosidase A (Europa Bioproducts).

After destaining, protein concentration was estimated using BCA assay (Thermo Fisher Scientific, 23225) and samples were in-gel digested with either ProAlanase prototype [1:50 (w/w), HCl pH 1.5, 2 h digestion at 37 °C] or with Trypsin (Sigma-Aldrich), rAsp-N (Promega, VA1160) and Glu-C (Sigma-Aldrich) [1:100 (w/w), 25 mm ammonium bicarbonate, pH 8.0, overnight at 37 °C]. For Glu-C a 50 mm potassium phosphate solution pH 8.0 was used, as a digestion buffer.
The activity of Trypsin, Asp-N and Glu-C was quenched by 1:10 acidification with 10% TFA (Sigma-Aldrich, T6508-500ML). For all enzymes, the resulting peptide mixtures were sequentially extracted from gel, by increasing % w/w of ACN. ACN was removed by vacuum centrifugation for 40 min at 60 °C using a SpeedVac^TM Concentrator (Thermo Fisher Scientific, Denmark) and peptide mixtures were loaded on an in-house packed StageTip C₁₈ cartridges.
Peptides were eluted in 40% ACN/0.1% FA. The organic phase was removed by vacuum centrifugation for 15 min at 60 °C in a SpeedVac^TM Concentrator (Thermo Fisher Scientific, Denmark). Samples were then diluted to approx. 0.1 µg/µL concentration with 0.1% TFA in 5% ACN.