2.5. Isobaric Tags or Relative and Absolute Quantitation (iTRAQ) Proteomics

The iTRAQ proteomic quantitative technique could use multiple (2-10) stable isotope tags to specifically label the amino groups of the peptides for tandem mass spectrometry analysis. It could compare the relative content of protein in different samples at the same time and could be used to study the difference of protein expression level in samples under different pathological conditions.

Three adrenal samples were randomly selected from each of the four groups. Next, protein extraction was performed using a vortex mixer (Haimen Qilin Bell Instrument Manufacturing Co. LTD, QL-901, Haimen, China) and an ultrasonic cell crusher (Nanjing Xianou Instrument Manufacturing Co. LTD, XO, Nanjing, China). The protein was reduced and sealed. After that, the protein was cut into peptides using trypsin, with an iTRAQ® kit (AB Sciex, PN: 4381664, CA, USA). The peptide mixtures in each group were labeled with different 8-plex iTRAQ reagents (AB Sciex, PN: 4381663, CA, USA). The labeled peptides in each sample were mixed in equal quantities. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) detection and analysis was performed with Durashell-C18, 4.6 mm × 250 mm, 5 μm, 100 Å (Agela, Item No: DC952505-0, Beijing, China). Protein analysis was carried out by Na-upgrade reversed-phase chromatography Q Exactive HF with a high-performance liquid chromatograph. The MS analysis of iTRAQ was performed using a Thermo Q Exactive HF-type mass spectrometer, and the original mass spectrometry files generated were processed by the supporting commercial software Proteome Discoverer 2.1 from Thermo.