Asp n

The UPS1 standard was purchased from Sigma-Aldrich (St. Louis, MO, USA) and contains 5 pmol per 50 partially truncated human proteins (see Supporting Information). The UPS1 proteins were resuspended in lysis buffer (8 M urea, 0.1 M ammonium bicarbonate, 0.1% RapiGest™ SF surfactant), reduced with 5 mM final concentration of TCEP (tris-2-carboxyethyl phosphine) at 37 °C for 10 min, and alkylated with 10 mM final concentration iodoacetamide at 25 °C for 30 min. Prior to enzymatic digestion, the buffer was adjusted to 2 M urea using 0.1 M ammonium bicarbonate. Each vial was digested at pH 8 and 37 °C with Asp-N (endoproteinase Asp-N 2 µg, sequencing grade, 11054589001; Roche Diagnostics, Rotkreuz, Switzerland) for 3 h. For Lys-C/Asp-N digest, a vial of UPS1 was first digested with Lys-C (endoproteinase Lys-C 5 µg, 11047825001; Roche Diagnostics) for 30 min at 37 °C, followed by the addition of Asp-N for 3 h at 37 °C. Following digestion, the reaction mixture was supplemented with buffer and enzymes as described above (yATE1 enzyme kinetics). Then, 90 min after adding yATE1, the reaction was quenched using 10% TFA to obtain a pH of 2 to 3 (as judged by pH paper). The peptides were purified by a standard C18 clean-up procedure12 (link) using a vacuum manifold (Sep-Pak Vac 1 cc (50 mg) tC18 cartridges, WAT054960; Waters Ltd, Elstree, UK).

To identify the C-mannosylation sites, we used the ultrasensitive Q-Exactive nanoLC-MS/MS system (Michalski et al., 2011 ). Purified Rspo1 samples were subjected to SDS–PAGE. After CBB staining, the visible band was excised and destained. In-gel digestion was performed using endoproteinase Asp-N (sequencing grade; Roche) and then trypsin (TPCK-treated; Worthington Biochemical, Worthington, OH). The digestion mixture was separated on a nanoflow LC (Easy nLC; Thermo Fisher Scientific, Waltham, MA) using a nano–electrospray ionization spray column (NTCC analytical column, C18, φ75 μm × 100 mm, 3 μm; Nikkyo Technos, Tokyo, Japan) with a linear gradient of 0–35% buffer B (100% acetonitrile and 0.1% formic acid) at a flow rate of 300 nl/min over 10 min, coupled on-line to a Q-Exactive mass spectrometer (Thermo Fisher Scientific) equipped with a nanospray ion source. MS and MS/MS data were acquired using the data-dependent top5 method. The resulting MS/MS data were searched against an in-house database, including the Rspo1 sequence, using MASCOT (Matrix Science, Boston, MA) with variable modifications: Gln → pyro-Glu (N-term Q), Oxidation (M), Propionamide (C), and Hex (W).