Peptides were analyzed using an online nano-LC-MS/MS system comprising an LTQ FT (Thermo Fisher Scientific), a hybrid linear ion trap and a 7-T Fourier transform ion cyclotron resonance mass spectrometer, coupled with an Ultimate 3000 Nano/Capillary LC System (Dionex). Samples were first loaded and desalted on a trap (0.3 mm inner diameter (i.d.) × 5 mm) at 20 μL/min with 0.1% formic acid for 5 min and then separated on an analytical column (75 μm i.d. × 15 cm) (both PepMap C18, LC Packings) over a 30-min linear gradient of 4-40% CH3CN, 0.1% formic acid for sample 1. The flow rate through the column was 300 nL/min. For sample 3, the separation gradient was a 120-min gradient 4-32% CH3CN/0.1% formic acid on a Atlantis C18 column (100 μm i.d. × 25 cm, Waters).
The LTQ FT mass spectrometer was operated in standard data-dependent acquisition mode controlled by Xcalibur 1.4 software. The survey scans were acquired on the FT-ICR (m/z 400-2000 for sample 1, or 400-1500 for sample 3) at a resolution of 100 000 at m/z 400, and one microscan was acquired per spectrum. For sample 1, the top three most abundant multiply charged ions with a minimal intensity at 1000 counts were subjected to MS/MS in the linear ion trap at an isolation width of 3 Th. For sample 3, the top 5 most abundant doubly and triply charged ions were subjected to MS/MS with the isolation width of 1.5 Th.
Precursor activation was performed with an activation time of 30 ms and activation Q at 0.25. The normalized collision energy was set at 35%. The dynamic exclusion width was set at 5 ppm with two repeats and a duration of 30 s for sample 1, 10 ppm with 1 repeats and duration of 60 s for sample 3. To achieve high mass accuracy, the automatic gain control target value was regulated at 4 × 105 (for sample 1) or 1 × 106 (for sample 3) for FT and 1 × 104 for the ion trap with a maximum injection time of 1000 ms for FT and 100 ms for the ion trap (sample 1) or 250 ms (sample 3). The instrument was externally calibrated using the standard calibration mixture of caffeine, a small peptide (sequence: MRFA) and Ultramark 1600.
The LTQ FT mass spectrometer was operated in standard data-dependent acquisition mode controlled by Xcalibur 1.4 software. The survey scans were acquired on the FT-ICR (m/z 400-2000 for sample 1, or 400-1500 for sample 3) at a resolution of 100 000 at m/z 400, and one microscan was acquired per spectrum. For sample 1, the top three most abundant multiply charged ions with a minimal intensity at 1000 counts were subjected to MS/MS in the linear ion trap at an isolation width of 3 Th. For sample 3, the top 5 most abundant doubly and triply charged ions were subjected to MS/MS with the isolation width of 1.5 Th.
Precursor activation was performed with an activation time of 30 ms and activation Q at 0.25. The normalized collision energy was set at 35%. The dynamic exclusion width was set at 5 ppm with two repeats and a duration of 30 s for sample 1, 10 ppm with 1 repeats and duration of 60 s for sample 3. To achieve high mass accuracy, the automatic gain control target value was regulated at 4 × 105 (for sample 1) or 1 × 106 (for sample 3) for FT and 1 × 104 for the ion trap with a maximum injection time of 1000 ms for FT and 100 ms for the ion trap (sample 1) or 250 ms (sample 3). The instrument was externally calibrated using the standard calibration mixture of caffeine, a small peptide (sequence: MRFA) and Ultramark 1600.
