4-Plex iTRAQ Based Quantitative Proteomic Analysis Using an Agilent Accurate -Mass Q-TOF
This document details a quantitative proteomic study using the Agilent 6520 Accurate-Mass Q-TOF LC/MS System coupled with the 1200 Series HPLC-Chip/MS System. The study utilized 4-plex iTRAQ labeling to identify and quantify differentially expressed proteins in brain tissue samples from patients with tuberculous meningitis compared to normal controls.
The analysis involved sample preparation, fractionation by strong cation exchange chromatography, and LC/MS/MS analysis. The high resolution of the Agilent 6520 system allowed for the minimization of interferences in the iTRAQ reporter ion region (m/z 114-117), enabling reliable quantitation. Data analysis was performed using the Spectrum Mill MS Proteomics Workbench suite.
| Enrichment column | 160 nL |
|---|---|
| Analytical pump flow | 300 nL/min |
| Loading pump flow | 4 µL/min |
| MS mode acquisition rate | 1 spectrum/sec |
| MS/MS mode acquisition rate | 3 spectra/sec |
| Drying gas flow | 5 L/min |
| Capillary voltage | 1900 V |
| Collision energy slope | 3 |
| Collision energy intercept | 2 |
Spectrum Mill software was used for protein identification and quantitation of iTRAQ reporter ion intensities.
Eight hundred proteins were identified from the LC/MS/MS analysis of the collected fractions.
Proteins were validated using a minimum peptide score of 6 and a protein score of 10 in the Spectrum Mill software.