Amniotic Fluid Proteome Profiling by iTRAQ

In this study, we used isobaric tag for relative and absolute quantitation (iTRAQ) gel-free proteomics to identify and quantify the proteins in amniotic fluid samples by referring to our previous studies^{20 (link),21 (link)}. In summary, we randomly selected 12 PTB AF samples and 12 FTB AF samples, followed by measuring the concentration of total protein. Then, per six AF samples with equal amounts of total protein were evenly pooled into one tube. As a result, two pooled PTB and two pooled FTB AF samples were acquired. Then, the four pooled protein samples were prepared with the standard protocol of the iTRAQ Reagents Multiplex Kit (4352135, Sciex). Next, the labeled samples passing the QC check were analyzed with LC/Q-Exactive Orbitrap MS (Thermo), followed by raw data analysis with Proteome Discoverer v2.4 (Thermo) using the MASCOT 2.5 database (Matrix Science). The detected protein abundance profiles were further analyzed with Partek to calculate p values (FTB vs. PTB).