Urinary proteome analysis of Coronary heart disease by iTRAQ approach


Coronary artery disease (CAD) is a manifestation of systemic atherosclerotic disease. It is assessed by intervention or traditional scoring risk factors. Diagnosis is limited by inaccurate and invasive methods. Developing noninvasive methods to screen for the risk of CAD is a major challenge. We aimed to identify urinary proteins associated with CAD. We utilized iTRAQ labeling followed by 2D LC-MS/MS to compare the urinary proteome of CAD patients to healthy cohorts. The multiple reaction monitoring (MRM) was used to verify the differential proteins. ROC analysis based on MRM data was used to evaluate the diagnostic application. A total of 876 proteins were quantified, and 100 differential proteins were found. Functional analysis revealed that the differential proteins were mainly associated with Liver X Receptor/Retinoid X Receptor (LXR/RXR) pathway activation, atherosclerosis signaling, production of nitric oxide and reactive oxygen species, and the top upstream regulator of the differential proteins by IPA analysis indicated to the APOE. Nineteen differential proteins were verified by MRM analysis. ROC based on MRM data revealed that the combination of two proteins (APOD and TFF1) could diagnose CAD with 85% sensitivity and 99% specificity (AUC 0.95). The urinary proteome might reflect the pathophysiological changes in CAD and be used for the clinical study of CAD.

A total of 44 individuals (22 CAD patients and 22 healthy controls) were included in this study.The 9 CAD samples as discovery group and the other 13 CAD samples as validation group. So did the control samples. First, we compared the pool sample from 9 CAD patients with that from 9 controls using iTRAQ. The differential proteins were screened. Then, we analyzed these differential proteins in validation groups using MRM.

CAD patients were found to have three aortic lesions by coronary angiography. The CON group showed negative results by coronary angiography. CAD patients included both sexes (68% male, 32% female), with an average age of 63 years. CONs were from unrelated subjects (56% male, 44% female), who had an average age of 57 years. The average BMI values of the CAD and CON groups were 26 and 25, respectively.

Protocol Description
Clinical materials

The midstream of the morning urine from CAD patients and CON individuals was used.

Sample preparation

The urinary samples (500 μg) were reduced with 20 mM DTT at 37 °C for 30 min, and alkylated with 50 mM iodoacetamide in darkness at room temperature for 45 min. The samples were loaded into a 10 kD ultracentrifugation tube and centrifuged at 14,000g,18°C for 30 min to deplete detrimental low-molecular-weight components.UA buffer (8 M urea in 0.1 M Tris/HCl, pH 8.5) and 25 mM NH4HCO3 was used to wash the samples. Trypsin was added to the urinary samples (1:50), which were digested using a microwave-assisted method [18]. The peptides were collected by centrifugation. The tryptic peptides were desalted using an SPE column according to the manufacturer’s instructions.

iTRAQ Or TMT labeling

Each sample was labeled with two iTRAQ reagents as follows: CON1 and CON2 received 114 and 116, respectively, and CAD1 and CAD2 received 115 and 117, respectively.


The iTRAQ-labeled digested mixture was fractioned with a high-pH RPLC column from Waters (4.6 mm× 250mm, C18, 3 μm). The samples were loaded onto the column in buffer A1 (100% H2O, pH 10). The eluted gradient was 5–90% buffer B1 (90% ACN; pH 10, flow rate, 1 mL/min) for 60 min. The flow peptides were collected per minute, and a total of 60 fractions were combined into 20 parts as follows: 1, 21, 41; 2, 22, 42; and so on.


Peptide fractions were further separated with an RP C18 self-packed capillary LC column (75 μm×100mm, C18, 3 μm). The eluted gradient was 5–30% buffer B2 (0.1% formic acid, 99.9% ACN; flow rate, 0.5 μL/min) for 40 min. Triple TOF 5600 was adopted to analyze the peptides. The MS data were acquired with the high sensitivity mode using the following parameters: 30 data-dependent MS/MS scans per full scan; acquisition of full scans at a resolution of 40,000 and MS/MS scans at 20,000; 35% normalized collision energy, charge state screening (including precursors with + 2 to + 4 charge states), and dynamic exclusion (exclusion duration 15 s); MS/MS scan range of 100–1800 m/z and scan time of 100 msec.

Data processing

The MS/MS spectra were searched against the human database (20,227 entry) from the UniProt website using the Mascot software (version 2.3.02). The Scaffold software (version 4.4.3) was used to further filter the database search results using the decoy database.