Acclaim pepmap c18 trap

Samples were dissolved in 0.1% TFA/2% acetonitrile (ACN) and analyzed in an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific, Waltham, MA, USA) equipped with a nanoelectrospray source and connected to an Ultimate 3000 RSLC nanoflow system (Thermo Scientific, Waltham, MA, USA). Peptides were loaded on an Acclaim PepMap C18 trap (Thermo Scientific, Waltham, MA, USA) and separated by a 50 cm µPAC™ (PharmaFluidics, Gent, Belgium) analytical column at 35 °C column temperature, utilizing 0.1% formic acid solvent as solvent A and 100% ACN with 0.1% formic acid as solvent B. We used a 120 min gradient at a flow rate of 500 nL/min, increasing it from 3.4% B to 21% B in 65 min, to 42% B in 32 min and to 75.6% B in 2 min; maintaining it for 3 min; then decreasing it to 3.6% B in 2 min; and maintaining it for 16 min. The spray voltage was set to 2 kV. A data-dependent acquisition method was used with a cycle time of 3 s and top N setting. Dynamic exclusion was set to 60 s, an AGC target of 4 × 10⁵, a maximum injection time of 50 msec and an Orbitrap resolution of 120,000 for MS1 scan. For all runs, an MS2 method was used with HCD fragmentation at 38%, first mass at 100 m/z, an MS² maximum injection time of 110 msec, an MS² isolation width of 0.8 m/z and an Orbitrap resolution of 60,000.

Sulfation analysis was done by in-gel digestion in preparation for LC-MS/MS. Briefly, Coomassie-stained light and heavy chain protein bands were excised from the SDS-PAGE gel and reduced with dithiothreitol (Fermentas, St. Leon-Rot, Germany). Following S-alkylation with iodoacetamide (Sigma-Aldrich Co., MO, USA) and digestion with porcine trypsin (Promega, WI, USA), fragments were eluted with 50% (v/v) acetonitrile/5% (v/v) formic acid (Sigma-Aldrich Co., MO, USA). Peptides were desalted and separated using an Acclaim PepMap C18 trap (75 μm × 2 cm) column (Thermo Fischer Scientific, MA, USA) and Acclaim PepMap C18 RSLC column (75 μm × 15 cm) (Thermo Fischer Scientific, MA, USA), respectively using a 4–60% (v/v) gradient of 80% (v/v) acetonitrile/0.1% (v/v) formic acid. Peptides were analysed using an AB Sciex (Miami, USA) 6600 TripleTOF MS, a triple Quadrupole Time of Flight (QTOF) Mass Spectrometer (MS). MS/MS scans were in the m/z range of 100 to 1800 Da. Data analysis was done using Protein Pilot (SCIEX, Canada) and Peaks v6^{32 (link)}.