Itraq reagents multiplex kit

To globally screen serum proteins, we randomly selected 12 age- and gender-matched FC and 12 KD serum samples. The selected serum samples were first subjected to high-abundance protein depletion with the Pierce Top 12 Abundant Protein Depletion Spin Columns (85165, Thermo). Then, the serum samples of six subjects were evenly pooled, resulting in two pooled FC and two pooled KD samples. The four pooled serum samples were then subjected to sample preparation with the iTRAQ Reagents Multiplex Kit (4352135, Sciex). After passing the standard QC check, the labeled serum samples were analyzed with LC/Q-Exactive Orbitrap MS (Thermo) for 24 h and the generated raw data was analyzed with Proteome Discoverer v2.4 (Thermo) by referring to the MASCOT 2.5 database (Matrix science). As a result, we acquired the relative abundances of detected proteins.

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).

The protein profiles were globally determined with iTRAQ gel-free proteomics and specifically validated with ELISA. We conducted the iTRAQ gel-free proteomics assay by referring to the previous study [18 (link)]. In summary, 24 serum samples from KD patients (12 with CAL and 12 without CAL) were first subjected to a high-abundant protein depletion with the Pierce Top 12 Abundant Protein Depletion Spin Columns (85165, Thermo, Waltham, MA, USA) to remove highly abundant proteins, including albumin, immuno-globulin (Ig)G, IgA, IgM, α1-acid glycoprotein, α1-antitrypsin, α2-macroglobulin, apolipoprotein A-I, apolipoprotein A-II, fibrinogen, haptoglobin, and transferrin. Next, the six serum samples from the KD patients with CAL were evenly pooled to generate one pooled serum library. Meanwhile, the six serum samples from KD patients without CAL were also evenly pooled to generate one pooled serum library. As a result, two pooled serum samples with CAL and two pooled serum samples without CAL were collected.
The four pooled serum libraries were subjected to peptide labeling with the iTRAQ Reagents Multiplex Kit (4352135, Sciex, Framingham, MA, USA) and analysis with a LC/Q-Exactive Orbitrap MS (Thermo, Waltham, MA, USA) for 24 h. By referring to the MASCOT 2.5 database (Matrix science), the relative abundances of detected proteins were determined.