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Table 4 Analytical condition of HPLC-API5000 or QTRAP5500 systems for peptide quantification

From: A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice

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.