Proline Quantification via aTRAQ and LC-MS/MS

To all tissue samples weighing 50 mg, 200 μL of methanol and the same amount of water (MiliQ) were added in a 1:1 ratio. Then, the tissues were homogenised and proline concentration was determined using the combination of the aTRAQ™ Kit for amino acid analysis (SCIEX, Framingham, MA, USA) and LC-MS/MS analysis. Samples for the aTRAQ™ Kit were prepared in accordance with the manufacturer’s protocol. LC-MS/MS analysis was performed using a 1260 Infinity HPLC instrument (Agilent Technologies, Santa Clara, CA, USA) and a 4000 QTRAP mass spectrometer (SCIEX, Framingham, MA, USA) equipped with an electrospray ionisation source. Sample analyses were performed in a random order. Detailed specifications of sample preparation and LC-MS/MS parameters have been described in our previous publications [48 (link),49 (link)]. Data acquisition and management were conducted using Analyst 1.5.2 software (Sciex, Framingham, MA, USA).