Mass spectrometer (API5000, QTRAP5500) | Turbo V ion source, SRM/MRM mode, total duration: 120 min. | |
Period 1 (100 min): | Positive ionization, CAD 12, CUR 40, GS1 20, GS2 40, IS 5500, TEM 500, ihe ON, EP 10, CXP 12. | |
Period 2 (20 min): | Negative ionization, CAD 12, CUR 40, GS1 20, GS2 40, IS -4500, TEM 500, ihe ON, EP -10, CXP -12. | |
HPLC (Agilent Technologies 1200 Series) | Column: | Waters XBridge BEH130 C18 (1.0 × 100 mm, 3.5 μm) column (Waters, Cat. No. 186003561) connected to a guard column (2.1 × 10 mm, 3.5 μm, Waters, Cat. No. 186003059) and a Sentry 2.1 × 10 mm guard holder (Waters, Cat. No. WAT097958). |
Mobile phases: | A and B consist of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. | |
Pump: | Peptides are separated and eluted from the column at 40°C (column oven) using a linear gradient with a 120 min run time at a flow rate of 50 μL/min. The sequence is as follows: | |
0 min: 99% A, 1% B | ||
5 min: 99% A, 1% B | ||
60 min: 40% A, 60% B | ||
61 min: 0% A, 100% B | ||
63 min: 0% A, 100% B | ||
65 min: 99% A, 1% B | ||
120 min: 99% A, 1% B | ||
Autosampler: | Injection volume, 40 μL. Sample loop, 100 μL. Temperature, 10°C. Needle wash with 0.1% formic acid in water for 5 sec. Well bottom sensing to inject the exact volume. | |
Valve (after HPLC column and before mass spec): | 0-20 min: directed to waste. | |
20-120 min: directed to mass spec ion source. |