Dionex ultimate 3000 nano hplc system

Analysis was conducted at the Centre for Proteomics and Genomic Research (CPGR) (Cape Town, South Africa). Nano-RP LC was performed on a Dionex Ultimate 3000 nano-HPLC system coupled with a Q-Extractive Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) for LC-MS/MS analysis. The mobile phase solvent system employed was solvent A: water/0.1% FA and solvent B: 100% ACN/0.1% FA. Solubilized peptides were loaded onto a C18 trap column (300 µm × 5 mm × 5 µm). Separation was performed on a C18 column (75 µm × 20 cm × 1.7 µm), and a linear gradient was generated at 300 nL/min with a change of 2–60% solvent B over 52 min. The mass spectrometer was operated in positive ion mode with a capillary temperature of 320 °C. The applied electrospray voltage was 1.95 kV. Lastly, mass spectrometry was performed using data-dependent acquisition MS/MS scans with a mass range of 350–2000 m/z.

LC-MS/MS was performed using an TIMS TOF Pro mass spectrometer (Bruker Daltonics, USA) combined with an UltiMate 3000 nano LC system (Thermo Fisher Scientific, MA, USA). The amount of loaded sample was 100 ng per injection. The tryptic peptide fraction (injection volume 1 μL) was analyzed in triplicate on a Dionex UltiMate 3000 Nano HPLC System (Thermo Fisher Scientific, MA, USA) coupled to a TiMS TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) using a captive spray ion source (positive ion mode, 1600 V) (Bruker Daltonics, Bremen, Germany). HPLC separation was performed on a C18 capillary column (25cm x 75μm 1.6μm) (Ion Optics, Parkville, Australia) by gradient elution. The mobile phase A was 0.1% formic acid diluted in water and mobile phase B was 0.1% formic acid diluted in acetonitrile. The separation was carried out by a 120-min gradient from 4% to 90% of phase B at a flow rate of 0.4 μL/min. Mass-spectrometric measurements were carried out using Parallel Accumulation Serial Fragmentation (PASEF) acquisition method. The measurements were carried out in m/z range from 100 to 1700. The range of ion mobility include a value from 0.60 – 1.60 Vs/cm2 (1/k0). The number of precursor ions per one PASEF cycle (1.2 sec) was 10. Low abundant precursor ions can be analyzed multiple times during PASEF cycle.

The identities of Coomassie-stained protein bands were independently determined by the Centre for Proteomic and Genomic Research (CPGR, Cape Town). Protein bands were recovered from the gel and fragmented by trypsin digestion, alongside a BSA reference standard. The resulting peptide solution was separated using the Dionex Ultimate 3,000 nano-HPLC system (ThermoFischer Scientific, USA) and then analyzed using a Q Exactive^™ Hybrid Quadrupole-Orbitrap Mass Spectrometer (ThermoFischer Scientific, USA). The spectra generated by LC-MS were analyzed with Byonic Software (Protein Metrics USA) using publically available sequences retrieved from UniProt (www.uniprot.org). Samples were interrogated against a merged database comprised of N. benthamiana, N. tabacum, Agrobacterium, and HIV proteomes.