Qstar xl mass spectrometer

The half of the protein was reduced with 10 mM DTT for 30 min at 60 °C and alkylated with 55 mM IAA for 30 min in the dark, following by digested with trypsin. The digested peptides were resuspended in 0.1% TFA and loaded onto Zorbax 300SB-C18 75 μm i.d. × 15 cm column via a trap column (Zorbax 300SB-C18 300 μm i.d. × 5 mm column). Peptides were then separated in an acetonitrile gradient (buffer A – 0.1% formic acid; buffer B – 100% acetonitrile and 0.1% formic acid) at a flow rate of 200 nl/min with an Agilent 1100 nanoHPLC system (Agilent, USA) and applied on-line to an Q Star XL mass spectrometer (AB Sciex, USA). The gradient was increased from 5% to 40% solution B over 110 min, followed by an increase to 95% B over 1 min, and then 95% B isocratic for 15 min. MS spectra were collected in full scan mode (350–1400 Da) followed by three MS/MS scans of the most intense ions.

The Agilent 1100 Series Nano HPLC interfaced to a QStar XL mass spectrometer (AB Sciex, Ontario, Canada) was used for analysis. Samples were loaded onto a ZORBAX 300SB-C18 trap column (5 µm, 300 Å, 0.3 mm) at a flow rate of 8 µL/min with 2% CH₃CN/0.1% trifluoroacetic acid and delivered to an Acclaim 300 (C18, 3 µm, 300 Å, 75 µm i.d. ×15 cm, Dionex Coorporation, CA) nanocolumn by a switching mechanism. Peptides were eluted from the nanocolumn at a flow rate of 250 nL/min with 2% CH₃CN/0.1% formic acid (solvent A) and 90% CH₃CN/0.1% formic acid (solvent B). The gradients used were: 0–30 min, 5% B (desalting); 30–80 min, 5–25% B; 80–95 min, 25–90% B; 95–110 min, 90% B; 110–120 min, 90–5% B; 120–130 min, 5% B.
A nanospray voltage in the range of 2000–2400 V was optimized daily. All nano LC MS/MS data were acquired in data-dependent acquisition mode in Analyst QS 1.1 (AB Sciex, Ontario, Canada). TOF MS survey scans with an m/z range of 300–1600 m/z for 1 s, followed by a product ion scan with an m/z range of 50–1600 m/z for 2 s each. Collision energy was automatically controlled by the IDA CE Parameters script.