3.5. Mass Spectrometry (MS)

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was carried out on a trapping nano-flow LC-Orbitrap MS system consisting of a Dionex Ultimate 3000 nano LC system, a Dionex Ultimate 3000 gradient micro-LC system with an WPS-3000 autosampler, and an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher Scientific, San Jose, CA, USA). For preparation of lyophilized LT samples, 50 μL 0.5% SDS was added to each sample, and samples were sonicated for 30 s and vortex-mixed for 10 min to reconstitute protein. Protein reduction and alkylation was performed sequentially by addition of 2 μL 200 μM DTT and 4 μL 500 μM iodoacetamide (IAM), each with 45 min incubation in a covered thermomixer under 37 °C with constant shaking. Protein was then precipitated by two-step addition of 60 and 300 μL chilled acetone, incubated at −20 °C for 3 h, and centrifuged for 30 min at 18,000× g under 4 °C to pellet precipitated protein. Pelleted protein was gently rinsed with 400 μL methanol, decanted, and wetted using 45 μL 50 mM Tris-FA. A volume of 5 μL trypsin dissolved in 50 mM Tris-FA (0.25 μg/μL) was added to each sample, and trypsinization was performed under 37 °C overnight (~16 h) with constant shaking in a covered thermomixer. Trypsinization was terminated by addition of 0.5 μL FA, then the derived peptide mixture was centrifuged at 18,000× g under 4 °C for 30 min, and supernatant was transferred to vials for analysis.

A single injection of derived peptides was analyzed for each sample. A large-i.d. trapping column (300 µm ID × 5 mm) was implemented prior to nano LC column (75-μm ID × 100 cm, and packed with 3 μm Pepmap C18) separation for high-capacity sample loading, matrix component removal, and selective peptide delivery. Mobile phase A and B were 0.1% FA in 2% acetonitrile and 0.1% FA in 88% acetonitrile. The 180 min LC gradient profile was 4–13% B for 15 min; 13% to 28% B for 110 min; 28% to 44% B for 5 min; 44% to 60% B for 5 min; 60% to 97% B for 1 min; and isocratic at 97% B for 17 min. MS was operated under data-dependent acquisition (DDA) mode, with a maximal duty cycle time of 3 s. MS1 spectra were acquired in the m/z range 400~1500 under 120 k resolution with dynamic exclusion settings (60 s ± 10 ppm). Precursor ions were filtered by quadrupole using a 1 Th wide window and fragmented by high-energy C-trap dissociation (HCD) at a normalized collision energy of 35%. MS2 spectra were acquired under 15 k resolution in either Orbitrap (OT) or Ion Trap (IT).