The peptide mixture was then fractionated on a surveyor HPLC system with a 1.7 μm Waters BEH C18 2.1 × 50 mm reversed-phase column (Waters Company, CA, United States). The chromatographic method was as follows: the peptides were eluted with a 5–35% B (A: 20 mM ammonium formate, pH = 10.0; B: acetonitrile) gradient for 16 min. Absorbance was measured at 214 nm, and a total of 40 fractions were collected and then combined into 10 fractions.
Itraq kit
The iTRAQ kit is a multiplexed quantitation kit designed for comparative proteomic analysis. The kit utilizes isobaric tags that are covalently attached to the N-terminus and lysine residues of peptides from different samples. The tagged peptides are then analyzed by mass spectrometry, allowing for the relative quantification of proteins across multiple samples simultaneously.
Lab products found in correlation
9 protocols using itraq kit
Quantitative Proteome Analysis via iTRAQ
The peptide mixture was then fractionated on a surveyor HPLC system with a 1.7 μm Waters BEH C18 2.1 × 50 mm reversed-phase column (Waters Company, CA, United States). The chromatographic method was as follows: the peptides were eluted with a 5–35% B (A: 20 mM ammonium formate, pH = 10.0; B: acetonitrile) gradient for 16 min. Absorbance was measured at 214 nm, and a total of 40 fractions were collected and then combined into 10 fractions.
Peptide Labeling with iTRAQ
Protein Digestion and Labeling Protocol
Quantitative Proteomics by 8-plex iTRAQ
The iTRAQ-labeled samples were mixed and resuspended in buffer A (2% acetonitrile, pH 10) and eluted with a 2–95% linear gradient of buffer B (90% acetonitrile, pH 10) over 90 min with a flow rate of 1 mL/min.
The samples were separated into 20 fractions on a C18 column with the HPLC system (Thermo Fisher Scientific) and analyzed using a Q Exactive Plus hybrid quadrupole-orbitrap Mass Spectrometer (Thermo Fisher Scientific). The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD023048.
Quantitative Proteomic Analysis of T2DM
Proteomic Analysis Workflow
Quantitative Proteomics of Infection Samples
After vacuum-dried, the iTRAQ-labeled peptides were reconstituted with 20 μl of 5 mM KH2PO4 containing 5% acetonitrile (pH 3.0), and separated via 2D-Liquid Chromatography (2D-LC) with an Ultimate Dual-gradient LC system (Dionex Ultimate3000).
HSYA Isolation and Analysis in C. tinctorius
Peptide Labeling and Fractionation for Mass Spec
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