Glu c

After the removal of supernatant, the resins with captured N-glycoproteins were diluted with 50 mM Tris + 1% SDS + 1% Triton (pH = 8). The glycoproteins on the resins were reduced with 20 mM dithiothreitol at 37°C for 2 h and carboxyamidomethylated with 40 mM iodoacetamide at room temperature for 40 min in the dark. Then 100 mM NH₄HCO₃, 80% ACN and 100 mM NH₄HCO₃ were sequentially added to wash the resins to remove non-specifically bound proteins. Trypsin, Trypsin & Glu-C, and chymoTrypsin were chose as proteases to digest the three batches of captured glycoproteins on the resins respectively. For Trypsin & Glu-C digestion, the two proteases were added into the sample together. The experimental procedures with each protease were all the same but different with the quantity of proteases added.
After the last wash of 100 mM NH₄HCO₃, each protease was added to digest the captured glycoproteins. The digest conditions of each protease were described as follows: Trypsin (Sigma-Aldrich, USA) was added with a weight ratio of Trypsin to protein at 1/25 and incubated at 37°C overnight; Trypsin & Glu-C were added with a weight ratio of Trypsin to protein at 1/25 and Glu-C (Roche, Germany) to protein at 1/20 and incubated at 37°C overnight; ChymoTrypsin (Sigma-Aldrich, USA) was added with a weight ratio of chymoTrypsin to protein at 1/10 and incubated at 37°C overnight.

Proteins were digested into peptides with trypsin (Promega, Madison, WI, USA) in 50 mM ammonium bicarbonate, pH 8.0 at 37˚C for 2 h (1:200 enzyme:substrate), with Glu-C (Roche Applied Science, Penzberg, Germany) in 25 mM ammonium carbonate, pH 7.8, at 24˚C for 18 h (1:20 enzyme:substrate), and with AspN (Roche Applied Science) and chymotrypsin (Roche Applied Science) in 100 mM Tris–HCl, 10 mM CaCl₂, pH 7.8, at 25˚C for 25 h (1:200 enzyme:substrate), as described previously [26 (link)]. Enzymatic digests were stopped by adding 10% trifluoroacetic acid (TFA) to a final pH <3. Peptides were then desalted with ZipTip C18 columns (Millipore) and lyophilized dry prior to analysis by ETD/CID-MS/MS.

30 μg purified ADAMTS17-PCD in Tris-HCl buffer containing 6 M urea was treated with 200 mM N-ethylmaleimide (NEM) to label solvent accessible free cysteinyl residues, then reduced with 200 mM DTT, and alkylated with 200 mM iodoacetamide prior to protease digestion. Mass-spectrometry grade trypsin, chymotrypsin, and GluC (Roche Diagnostics, Indianapolis, IN) were added at a 1:20 (protease to protein) ratio, and the digest was incubated overnight at room temperature for completion. The protein digest was desalted using C18 SPE method (PepClean C-18 spin column) and reconstituted in 30 μL 1% acetic acid for LC-MS/MS analysis as described above. The identification of cysteine-containing ADAMTS17 peptides was performed by searching the MS/MS spectra specifically against the sequence of ADAMTS17 using the program Sequest (bundled into Proteome Discoverer v. 1.4.0) considering the formation of both carbamidomethylated and NEM modified cysteine residues. All cysteine-containing peptides were high confidant identifications and were subjected to manual validation.