Orbitrap exploris 480 ms

Peptide mapping was performed to help verify the presence of identified VP PTMs, VP variants and VP fragments. Digestion of each empty and full capsid sample for peptide mapping was performed using a SMART Digest™ magnetic bead bulk pepsin kit (Thermo Scientific) and followed a slightly modified version of the protocol previously described by Guapo et al. [9 (link)] (Supplementary Information (SI) 1). Peptide mapping was performed in technical triplicate using a Vanquish Neo Ultra-High-Performance Liquid Chromatography (UHPLC) platform coupled to an Orbitrap Exploris 480 MS (Thermo Scientific, Bremen, Germany) following a modified version of the procedure described in Guapo et al. [9 (link)] (SI 2). Peptide identification and relative PTM quantitation was performed using BioPharma Finder™ (BPF) Version 5.1 (Thermo Scientific, San Jose, CA, USA) (SI 3 and Table S2).

The protein samples from trace IA and STA tissue were analyzed on an UltiMate 3000 nanosystem (Thermo Fisher Scientific, USA) connected to an Orbitrap Exploris 480 MS in combination with Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS). The peptides were separated on a 75 μm × 25 cm long column (2 μm id) at a flow rate of 300 nl/min for 150 min. The data of TMT‐labeled serum peptides were acquired on an easyNano system (Thermo Fisher Scientific, USA) with a 75 μm × 25 cm long column (2 μm id) connected to Orbitrap Fusion Tribrid Mass Spectrometer (MS) (Thermo Fisher Scientific, USA) by a 120 min LC separation.

All samples were analyzed on the UltiMate3000 system (Thermo Fisher Scientific) using an integrated monolithic C18 capillary column (ID, 75 µm, Length, 50 cm, Uritech, Beijing) and maintained at a constant column temperature of 60 °C throughout the entire experiment. Separation was achieved with stepped linear solvent gradients, all performed at a fixed flow rate of 1.5 µl/min with durations of 25 min. The organic modifier content (acetonitrile acidified with 0.1% v/v formic acid) was first increased from 5 to 20% in 15.5 min and then increased from 20 to 30% in 5 min. Then, it was increased from 30 to 50% in 1 min and increased to 90% in 0.1 min. Next, it was maintained for 1.3 min. Finally, it was increased from 90 to 5% in 0.1 min and maintained for 2 min.
The mass spectrometer was operated in positive mode with the Orbitrap Exploris 480 MS (Thermo Fisher Scientific). DIA was performed using the following parameters. For the full scan, the resolution was set at 120,000 with a normalized AGC target of 300%. The maximum injection time was set to 50 ms, and the scan range was from 350 to 1200 m/z. For DIA scans, the resolution was set at 30,000 with a normalized AGC target of 200% and a maximum injection time of 50 ms. A normalized HCD collision energy of 30% was used.

LC-MS/MS analysis samples were analyzed by the commercial company MS-Omics. The analysis was carried out using a UPLC system (Vanquish, Thermo Fisher Scientific) coupled with a high-resolution quadrupole-orbitrap mass spectrometer (Orbitrap Exploris 480 MS, Thermo Fisher Scientific) using an electrospray ionization interface operated in positive ionization mode. The UPLC was performed using a slightly modified version of the protocol described (Hsiao et al., 2018 (link)). Data were manually inspected to generate MS/MS spectra using Freestyle 1.4 (Thermo Fisher Scientific).