Symmetry c18 nanoacquity trap column

For Ig-bound protein analysis, protein digestion and identification by LC-MS/MS was performed using our established protocol [19 (link),45 (link),46 (link)]. Briefly, a nanoAcquity UPLC system coupled in-line with WATERS SYNAPT G2-Si mass spectrometer was used for the separation of pooled digested protein fractions. The system was equipped with a Waters Symmetry C18 nanoAcquity trap-column (180 μm × 20 mm, 5 μm) and a Waters HSS-T3 C18 nanoAcquity analytical column (75 μm × 150 mm, 1.8 μm). Data were acquired in resolution mode with SYNAPT G2-Si using Waters Masslynx (version 4.1, SCN 851). The mass spectrometer was operated in V-mode with a typical resolving power of at least 20,000. All analyses were performed using positive mode ESI using a NanoLockSpray source. The lock mass channel was sampled every 60 s. Accurate mass LC-HDMSE data were collected in an alternating, low energy (MS) and high energy (MSE) mode of acquisition with mass scan range from m/z 50 to 1800. The spectral acquisition time in each mode was 1.0 s with a 0.1 s inter-scan delay. The acquired LC-HDMSE data were processed and searched against protein knowledge database (Uniprot and TruEMBL, 92,355 human protein sequences) through ProteinLynx Global Server (Version 3.0.2, Waters Company) with 4% FDR.