Lc q exactive orbitrap ms

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.

In this study, isobaric tagging for relative and absolute quantification (iTRAQ) gel-free proteomics was used to identify salivary protein biomarkers. To screen salivary protein biomarkers, we randomly selected saliva samples from age-matched Streptococcus pneumoniae (S. pneumoniae) infection and influenza A-associated pneumonia cases for proteomic analysis. The selected saliva samples were subjected to high-abundance protein depletion using Pierce Top 12 Abundant Protein Depletion Spin Columns (85,165; Thermo Fisher Scientific, Waltham, MA, USA). Salivary samples from each of the three subjects were combined to obtain a combined pneumonia sample from Streptococcus pneumoniae and Type A influenza. Salivary samples were labeled and prepared using the iTRAQ Reagents Multiplex Kit (435,2135, AB SCIEX, Foster City, CA, USA). After standard quality control inspection, the labeled salivary samples were analyzed using LC/Q-Exactive Orbitrap MS (Thermo) and interpreted with Proteome Discoverer v2.4 (Thermo) referring to the MASCOT 2.5 database to obtain the relative abundance of proteins.

iTRAQ gel-free proteomics were selected to globally screen and compare the protein contents of exosomes derived from AD-MSCs and WJ-MSCs, as reported previously [48 (link)]. In brief, the collected exosome protein samples were first subjected to high-abundance protein depletion with the Pierce Top 12 Abundant Protein Depletion Spin Columns (85165; Thermo Fisher Scientific). Then, the protein samples from AD-MSCs and WJ-MSCs were prepared using the iTRAQ Reagents Multiplex Kit (4352135; Sciex, Framingham, MA, USA). After a standard quality check, labeled peptide samples were determined using LC/Q-Exactive Orbitrap MS (Thermo Fisher Scientific) and analyzed using Proteome Discoverer v2.4 (Thermo Fisher Scientific) with reference to the MASCOT 2.5 database (Matrix science, Portland, OR, USA) to determine the relative abundances of the detected proteins.

To globally screen proteins in tear fluid, we conducted TMT gel-free proteomics assay. First of all, we selected ten tear fluid samples and had them subjected to protein digestion by trypsin enzyme to generate peptides. Then, the ten peptide samples were subjected to peptide labeling with the TMT 10-plex Reagents Kit (90111, Thermo Fisher, Waltham, MA, USA) by referring to the manufacturer’s protocols. Next, the labeled peptide samples, passing the standard QC check, were analyzed with LC/Q-Exactive Orbitrap MS (Thermo Fisher) for 24 h. The generated raw data was further analyzed with Proteome Discoverer v2.4 (Thermo Fisher) by referring to the MASCOT 2.5 database (Matrix science). By doing so, the relative abundances of proteins in tear fluid were detected. The proteins, with false discovery rates less than 1% and with at least two unique peptides, were considered. In addition, median normalization was applied to normalized the protein abundances among samples for reducing the system error which may be contributed by sample preparation.