Itraq 4plex

Manufactured by AB Sciex

Sourced in United States

The iTRAQ 4plex is a labeling reagent kit designed for quantitative proteomic analysis. It enables the simultaneous identification and relative quantification of proteins across up to four samples in a single mass spectrometry experiment.

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Lab products found in correlation

Ettan lc system, by GE Healthcare (1 mentions) Agilent 1100 hplc system, by Agilent Technologies (1 mentions) Tmt 6plex, by Thermo Fisher Scientific (1 mentions) Sep pak plus c18 cartridge, by Waters Corporation (1 mentions)

6 protocols using itraq 4plex

Multiplexed Protein Quantification by iTRAQ

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Isobaric tags for relative and absolute quantification (iTRAQ) analysis
was performed using 10 μg of in-solution digested sample from
each patient and control. Samples were labeled with iTRAQ 4plex (AB
SCIEX, Framingham, MA, USA) according to the manufacturer’s
protocol, resulting in a total of five sets of 4plex experiments (Figure 1). A mixed sample containing 5 μg of sample
from each of the patients and controls was labeled with the iTRAQ
114-tag in all five experiments and used to normalize between experiments.
Each 4plex experiment was diluted 10 times in buffer A (0.5% formic
acid and 5% acetonitrile) and separated on a Higgins Analytical strong
cation exchange (SCX) column (PL-SCX 1000 Å 5 μm 20 ×
2.1 mm column, Higgins Analytical, Rengstorff, CA, USA) equilibrated
in buffer A and connected to an Ettan LC system (GE Healthcare, Wauwatosa,
WI, USA). The peptides were eluted using a 100 μL/min flow rate
and a 1% B/min linear gradient from buffer A to buffer B (buffer B
containing 1 M NaCl). The 15 collected fractions were lyophilized,
redissolved in 100 μL 0.1% formic acid, and desalted using POROS
50 R2 material packed in GELoader tips. The desalted samples were
lyophilized and resuspended in 12 μL 0.1% formic acid and stored
at −20 °C.

Poulsen E.T., Dyrlund T.F., Runager K., Scavenius C., Krogager T.P., Højrup P., Thøgersen I.B., Sanggaard K.W., Vorum H., Hjortdal J, & Enghild J.J. (2014). Proteomics of Fuchs’ Endothelial Corneal Dystrophy Support That the Extracellular Matrix of Descemet’s Membrane Is Disordered. Journal of Proteome Research, 13(11), 4659-4667.

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Quantitative Peptide Labeling with iTRAQ

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Peptide labeling with iTRAQ 4plex (AB Sciex, Framingham, MA) was performed according to the manufacturer’s protocol. For each analysis, 100 μg of protein from each cell line was labeled with one tube of iTRAQ 4plex reagent. The 11-18 and 11-18R cells were labeled with the iTRAQ 4-plex as follows: 114- and 116-channels, 11-18 cells; 115- and 117-channels 11-18R cells. Lyophilized samples were dissolved in 60 μL of 500 mM triethylammonium bicarbonate, pH 8.5, and the iTRAQ reagent was dissolved in 70 μL of isopropanol. The solution containing peptides and iTRAQ reagent was vortex mixed and then incubated at room temperature for 1h and concentrated to 40 μL under vacuum. Samples labeled with the four different isotopomeric iTRAQ reagents were combined and evaporated to dryness. Peptides then were dissolved in 3% aqueous acetonitrile containing 0.1% formic acid solution before micro-bRPLC fractionation.

Lee H.J., Kim H.J, & Liebler D.C. (2016). Efficient micro-scale basic reverse phase peptide fractionation for global and targeted proteomics. Journal of proteome research, 15(7), 2346-2354.

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Comparative Proteomic Analysis of Alexandrium Species

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For each biological replicate, one technical replicate was provided for iTRAQ analysis. Protein samples (50 µg) were reduced using DTT, alkylated with iodoacetamide and digested with trypsin. Tryptic digests were desalted using C18 zip-tip prior to iTRAQ 4-plex (AB Sciex, Foster City, CA, USA) labeling. A. minutum was labeled with 114 and 117 isobaric tags while A. tamutum with 115 and 116 isobaric tags. Labeled samples were combined in a 1:1:1:1 ratio (with BSA as internal control) and prefractionated offline using strong cation exchange column, polysulfoethyl A (200 × 2.1 mm; 5 µm) (PolyLC INC., Columbia, MD, USA) column, on an Agilent 1100- HPLC system (Agilent Technologies, Palo Alto, CA, USA). Solvent A was: 25% CH₃CN; 0.1% HCOOH while Solvent B was 25% CH₃CN; 0.1% HCOOH; 500 mM KCl. A linear gradient from 0–100% Solvent B in 45 min was performed with flow rate set at 200 µL/min. Peptides were detected using a UV detector set at λ = 214, 260 and 280 nm. A total of 21 fractions were collected using a fraction collector. Fractions were further analyzed using High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS).

Subong B.J., Lluisma A.O., Azanza R.V, & Salvador-Reyes L.A. (2020). Differentiating Two Closely Related Alexandrium Species Using Comparative Quantitative Proteomics. Toxins, 13(1), 7.

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Isobaric Labeling Protocol for IP Samples

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Starting with IP samples on beads supplied in 100 μL of 100 mM TEAB buffer, reduction and alkylation of disulfide bonds were carried out by addition of 2 μL of 50 mM Tris(2-carboxyethyl) phosphine (TCEP) in 100 mM TEAB (Sciex 4326685) for 60 min at 60 °C, followed by addition of 1 μL of 2% S-methyl methanethiosulphonate in isopropanol (Sciex 4352159) for 10 min at room temperature. Proteins in this solution were then digested by the addition of 250 ng of TPCK-treated trypsin in 50 mM TEAB (Sciex 4352157) and overnight incubation at 37 °C with gentle shaking. iTRAQ4-plex (Sciex) or TMT 6-plex (Thermo Fisher Scientific) reagents were resuspended in 50 µL isopropanol and added to each sample followed by vortex and spin; the specific iTRAQ or TMT labels used for each pair of bait or control IP replicates are indicated in Supplementary Data 6. The samples were combined and incubated at room temperature for 2 h, and then washed, extracted, and concentrated by solid phase extraction using Waters Sep-Pak Plus C18 cartridges. Organic solvent was removed, and the volumes were reduced to 80 μL via speed vacuum.

Zhu Q.M., Hsu Y.H., Lassen F.H., MacDonald B.T., Stead S., Malolepsza E., Kim A., Li T., Mizoguchi T., Schenone M., Guzman G., Tanenbaum B., Fornelos N., Carr S.A., Gupta R.M., Ellinor P.T, & Lage K. (2024). Protein interaction networks in the vasculature prioritize genes and pathways underlying coronary artery disease. Communications Biology, 7, 87.

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Quantitative Proteomics of A. fumigatus under Oxidative Stress

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Three biological replicates of A. fumigatus Af293 mycelium samples from four different growth conditions were measured as described above in the section “Strain and growth conditions”. These included iron-replete conditions with (+Fe/+H₂O₂) and without (+Fe/-H₂O₂) oxidative stress induced by 3 mM hydrogen peroxide, as well as iron-deprived conditions with (-Fe/+H₂O₂) and without (-Fe/-H₂O₂) oxidative stress. Proteins were isolated and digested as described previously [52 (link)]. Labeling of tryptic peptides with iTRAQ 4-plex (Sciex, Darmstadt, Germany) reagents was performed according to the manufacturer’s manual. Each biological replicate was represented in one 4-plex reaction. The three 4-plex reactions were made up as follows: 114 (+Fe #1; -Fe/+H₂O₂ #2; +Fe/+H₂O₂ #3), 115 (-Fe #1; +Fe #2; -Fe/+H₂O₂ #3), 116 (+Fe/+H₂O₂ #1; -Fe #2; +Fe #3), 117 (-Fe/+H₂O₂ #1; +Fe/+H₂O₂ #2; -Fe #3). Each 4-plex reaction was combined, dried (speed vac), and resolubilized in 40 μL of 0.05% (v/v) trifluoroacetic acid in 2/98 (v/v) acetonitrile (ACN)/H₂O for LC-MS/MS analysis.

Kurucz V., Krüger T., Antal K., Dietl A.M., Haas H., Pócsi I., Kniemeyer O, & Emri T. (2018). Additional oxidative stress reroutes the global response of Aspergillus fumigatus to iron depletion. BMC Genomics, 19, 357.

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Peptide Labeling and Phosphopeptide Enrichment

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Peptides (100 μg per experimental condition) were labeled with iTRAQ 4 plex (Sciex) reagent according to the manufacturer's specifications. Samples were combined in 1:1:1:1 ratio and dried down. For phosphopeptide enrichment, we used the TiSH method combining TiO 2 -SIMAC-TiO 2 and pre-fractionation with HILIC [25] . Offline pre-fractionation using HILIC was performed as reported previously [26] .

da Silva F.A., Rodrigues-Ribeiro L., Melo-Braga M.N., Passos-Silva D.G., Sampaio W.O., Gorshkov V., Kjeldsen F., Verano-Braga T, & Santos R.A.S. (2022). Phosphoproteomic studies of alamandine signaling in CHO-MrgD and human pancreatic carcinoma cells: An antiproliferative effect is unveiled. Proteomics, 22(17).

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