Easy nlc nano uplc

Extracted peptides were separated at a 300 nL/min flow rate using a reversed‐phase column (100 μm × 150 mm, 3 μm ReproSil‐Pur 120 C18‐AQ, 1.9 μm, Dr Math), followed by analysing with the EASY‐nLC nano‐UPLC (Thermo Fisher Scientific, Bremen, Germany). It performed at a 120‐min gradient elution with the UPLC mobile phase A [formic acid (FA) (0.1%) with acetonitrile (ACN) (2%)] and B [ACN (80%) with FA (0.1%)] (8%–30% B for 92 min, 30%–40% B for 20 min, 40%–100% B for 2 min, 100% B for 2 min, 100%–2% B for 2 min and finally 2% B for 2 min). Q‐Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) applying the DDA (data‐dependent acquisition) strategy which MS for the 20 most intense ions using a normalized collision energy of 27% for HCD and an isolation window of 2 m/z were used to analyse the peptides. Resolution of 70,000 for MS1 (at 200 m/z) and 17,500 for MS2 in an orbitrap analyser were processed in the following analyses. The automatic gain control target for MS1 and MS2 were set to 3.0 E⁺⁶ with max IT 50 ms and 5.0 E⁺⁴ with max IT 100 ms respectively. Thirty seconds were set for dynamic exclusion.

Extracted peptides were separated using a reversed‐phase column (100 μm × 150 mm, 3 μm ReproSil‐Pur 120 C18‐AQ, 1.9 μm, Dr Math) at a 300 nL/min flow rate and analysed with the EASY‐nLC nano‐UPLC (Thermo Fisher Scientific, Bremen, Germany). The UPLC mobile phase A [0.1% formic acid (FA) with 2% acetonitrile (ACN)] and B [80% ACN with 0.1% FA] were used to perform a 120‐min gradient elution as follows: 8%‐30% B for 92 minutes, 30%‐40% B for 20 minutes, 40%‐100% B for 2 minutes, 100% B for 2 minutes, 100%‐2% B for 2 minutes and finally 2% B for 2 minutes. Peptides were analysed using a Q‐Extractive mass spectrometer (Thermo Fish Scientific, Bremen, Germany) coupled with MS for the 20 highest‐intensity ions with a normalized collision energy of 27% for HCD and an isolation window of 2 m/z. The full mass and subsequent MS/MS analyses were performed in an Orbitrap analyzer with a resolution of 70 000 for MS1 (at 200 m/z) and 17 500 for MS2, respectively. The automatic gain control target for MS1 was set to 3.0 E⁺⁶ with max IT 50 ms and 5.0 E⁺⁴ for MS2 with max IT 100 ms. The top 20 most intense ions were fragmented by HCD with normalized collision energy of 27% and isolation window of 2 m/z. The dynamic exclusion was set at 30 seconds.