Easy nlc1200 nano uplc liquid phase system

Two microliters of total peptide were taken from each sample and separated with an EASY-nLC1200 nano-UPLC Liquid Phase System (Thermo Fisher Scientific). Data were collected using a mass spectrometer (Q-Exactive HFX; Thermo Fisher Scientific) equipped with a nano-electrospray ion source. Separation was performed with a reversed-phase column (100 μm ID × 15 cm, Reprosil-Pur 120 C18-AQ, 1.9 μm). The mobile phase adopted the acetonitrile–water-formic acid system, in which mobile phase A was 0.1% formic acid-98% aqueous solution (2% in acetonitrile) and phase B was 0.1% formic acid-80% acetonitrile (20% in water). After the column was equilibrated with a 100% A phase, the sample was directly loaded onto the column and passed through the column ladder degree separation with a flow rate of 300 nL/min and gradient length of 120 min. Mobile phase B ratios were applied as follows: 5% for 2 min, 5%–22% for 98 min, 22%–45% for 16 min, 45%–95% for 2 min, and 95% for 2 min. Mass spectrometry analysis used a data-dependent acquisition mode with a total analysis time of 120 min and positive ion detection mode.

Proteomic detection of rat carotid arteries was performed by Aksomics (Shanghai, China). Rat carotid arteries were lysed with RIPA lysate and protease inhibitors, and protein concentration was measured. Peptide samples were obtained by acetone precipitation, protein redissolution, reduction, alkylation, and trypsin digestion. Peptide samples were desalted and dried under vacuum. Peptides were redissolved to 1 µg/µl in buffer. 2 µg of peptide from each sample was separated on an EASY-nLC1200 nano-UPLC liquid phase system (Thermo Fisher Scientific), detected by a Q-EXactive mass spectrometer (Thermo Fisher Scientific), and analyzed on a 100 μm ID × 15 cm reversed-phase chromatography column (Reprosil-Pur 120 C18-AQ, 1.9 μm, Dr. Math). The sample flow rate through the chromatography column was 300 nL/min and the gradient time was 120 min.
The acquired raw files were processed using MaxQuant (1.5.6.0). The protein database was obtained from the UNIPROT database (Uniprot_rat_2018_10). The type of quantification was label-free quantification (LFQ). Three groups of biological replicates were set up in the experiment, and Student’s t-test was performed. Proteins with expression fold change (ratio A/B) > 1.25, P value < 0.05 were defined as significantly different.