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Agilent High-Throughput Protein Quantitation Using MRM Application Note
This document describes a workflow for quantitative proteomics and biomarker validation using Multiple Reaction Monitoring (MRM) on Agilent mass spectrometers. The study utilizes a triple quadrupole (Q0Q) mass spectrometer interfaced with an HPLC-Chip for nanoflow LC/MS analysis to achieve high sensitivity and selectivity.
The workflow involves sample preparation of human plasma, depletion of abundant proteins, and digestion. Data acquisition is performed using dynamic MRM functionality in MassHunter software, allowing thousands of peptide transitions to be monitored in a single run. Results are analyzed using Mass Profiler Pro for statistical analysis, including principal component analysis and hierarchical clustering.
Agilent 6520 Accurate-Mass Q-TOF specifications
| Analytical column dimensions | 150 x 0.075 mm |
|---|---|
| Enrichment column volume | 40 nL |
| Injection volume | 1 pL |
| Analytical pump flow rate | 300 nL/min |
| Loading pump flow rate | 3 L/min |
| Drying gas flow | 5 L/min |
| Capillary voltage | 1,800 V |
| Collision energy slope | 3.6 |
| MS response RSD | below 5% |
| Retention time RSD | less than 0.04 min |
Source: Agilent High-Throughput Protein Quantitation Using MRM Application Note
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Questions answered in these manuals
What software is used for dynamic MRM acquisition?
MassHunter acquisition software equipped with dynamic MRM functionality is used to monitor the transition list.
From: Agilent High-Throughput Protein Quantitation Using MRM Application Note
Which software is used for statistical analysis of MRM results?
Mass Profiler Pro is used to perform principal component analysis, hierarchical clustering, and ANOVA analysis.
From: Agilent High-Throughput Protein Quantitation Using MRM Application Note
What software is used for data analysis?
Data was processed using Agilent MassHunter Qualitative Analysis software, Agilent MassHunter BioConfirm and Agilent Spectrum Mill MS Proteomics Workbench software packages.
From: Agilent 6520 Accurate-Mass Q-TOF LC/MS Peptide Mapping Application Note
What is the flow rate for the enrichment column?
The flow rate from the Agilent 1200 Series Capillary Pump to the enrichment column is 3 L/min.
From: Agilent 6520 Accurate-Mass Q-TOF LC/MS Peptide Mapping Application Note