Discovery- and target-based protein quantification using iTRAQ and pulsed Q collision induced dissociation (PQD)

Area of Science:

• Proteomics
• Mass Spectrometry
• Quantitative Biology

Background:

• Pulsed Q collision-induced dissociation (PQD) aids reporter ion detection for quantitative proteomics on Linear Trap Quadrupole (LTQ) platforms.
• Reproducibility issues with PQD reporter ion ratios limit its application, especially for low-abundance proteins.
• Existing optimizations for PQD in iTRAQ quantification on LTQ have not established a universally applicable strategy.

Purpose of the Study:

• To develop an improved PQD strategy for reproducible iTRAQ-based quantitative proteomics on LTQ instruments.
• To address the limitations of discovery-based approaches in achieving reliable quantification of low-abundance proteins.
• To provide a viable alternative for researchers lacking access to Orbitrap or similar high-end mass spectrometers.

Main Methods:

• Implemented a target-based data acquisition strategy, bypassing data dependency for repetitive measurements across chromatographic peaks.
• Utilized cAMP treatment in S49 cell lysates for validation of differentially expressed proteins.
• Compared the target-based PQD approach with higher-energy collisional dissociation (HCD) on an Orbitrap mass spectrometer.

Main Results:

• The target-based approach significantly improved the reproducibility of reporter ion ratios compared to discovery-based PQD.
• Successfully validated differentially expressed proteins identified through discovery-based PQD.
• Achieved quantitative results comparable to those obtained using HCD on an Orbitrap.

Conclusions:

• The target-based PQD strategy offers a robust and reproducible method for iTRAQ quantitative proteomics on LTQ platforms.
• This approach enhances the ability to quantify low-abundance proteins, overcoming previous limitations.
• LTQ users can effectively perform quantitative proteomics without requiring more expensive instrumentation.