Quantitative proteomics using multiple reaction monitoring (MRM) has emerged as an important methodology for biomarker validation. Multiple-reaction monitoring on a triple quadruple (QQQ) mass spectrometer provides superior sensitivity and selec- tivity for targeted peptides in a complex sample. MRM also offers high precision in quantitation and a fast scan speed, which makes it an ideal technology for validating biomarkers in a high-throughput fashion. This application note demonstrates the entire workflow for biomarker validation offered by Agilent Technologies using multiple reaction monitoring together with statistical analysis software. Thousands of peptide transitions were generated using Agilent Q-TOF and Spectrum Mill MRM selector. The transition list was imported into MassHunter acquisition software equipped with dynamic MRM functionality and monitored in a single LC/MS run. Finally, MRM quantitation results were analyzed using Mass Profiler Pro for principle component analysis (PCA), hierarchical clustering and ANOVA analysis.
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.
| 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 |
MassHunter acquisition software equipped with dynamic MRM functionality is used to monitor the transition list.
Mass Profiler Pro is used to perform principal component analysis, hierarchical clustering, and ANOVA analysis.
The cycle time is set to obtain at least 15 data points across the peaks.