Agilent AssayMAP Bravo Technology Enables Reproducible Automated Phosphopeptide Enrichment from Complex Mixtures Using High‑Capacity Fe(III)‑NTA Cartridges

Význam tématu

Advances in phosphoproteomics demand robust and reproducible workflows for selective enrichment of phosphopeptides prior to LC/MS. Immobilized metal affinity chromatography (IMAC) with Fe(III)-NTA resin is a leading technique but suffers from variability due to manual handling and suboptimal chemistry. Automating this process enhances throughput, consistency, and scalability, enabling reliable analysis of cellular phosphorylation dynamics.

Cíle a přehled studie / článku

The study evaluates the performance of the Agilent AssayMAP Bravo platform with high-capacity Fe(III)-NTA cartridges for automated phosphopeptide enrichment. Key objectives include: optimizing solution chemistry (acid type/concentration, percent organic solvent), determining cartridge binding capacity for model and complex samples, assessing sequence-dependent biases and sample-loading flow rates, and demonstrating reproducibility with yeast whole-cell digests.

Použitá metodika a instrumentace

• Sample preparation: In-solution digestion of bovine α-casein and yeast lysates; reduction, alkylation, trypsin digestion, C18 desalting, lyophilization.
• Automated enrichment: Agilent AssayMAP Bravo using Phosphopeptide Enrichment v2.0 App and 5-µL Fe(III)-NTA cartridges; priming/equilibration in ACN/TFA; loading, wash, and elution with aqueous ammonia.
• Optimization variables: acid type (TFA vs. acetic acid), acid concentration (0.01–1.0%), % ACN (60–90%), sample-loading flow rate (2.0–6.5 µL/min), cartridge capacity (25–400 µg α-casein; 200–1,200 µg yeast digest).
• Instrumentation: Agilent 1290 Infinity Binary LC with AdvanceBio C18 columns; Agilent 6550 iFunnel Q-TOF MS with Dual Jet Stream ESI; data processed with Spectrum Mill and Skyline.

Hlavní výsledky a diskuse

• α-Casein model: 31 phosphopeptides enriched with >92% specificity; complete elution in <10 µL; global capacity ~150 µg digest (~60 nmol phosphate); sequence analysis revealed that acidic phosphopeptides maintain binding capacity better than highly basic variants upon cartridge saturation.
• Chemistry optimization (yeast digest): 0.1% TFA with 80% ACN in both sample and wash achieved ~2,300 distinct phosphopeptides and >96% selectivity; comparable performance with 0.1% acetic acid.
• Sample-loading flow rate: Optimal at 3.5–5.0 µL/min; higher rates (>5 µL/min) reduced both identifications and selectivity due to insufficient residence time for binding.
• Cartridge capacity for yeast: 1st pass enrichment plateaued above 800 µg load; a 2nd pass recovered additional acidic phosphopeptides but with diminishing returns; capacity considerations critical to avoid bias.
• Reproducibility: Four technical replicates of 500 µg yeast digest yielded on average 2,515 distinct phosphopeptides (CV 4.7%) with 96.2% selectivity (CV 2.6%), representing a 23-fold increase in identifications versus unenriched controls.

Přínosy a praktické využití metody

Automated phosphopeptide enrichment on the AssayMAP Bravo platform delivers high selectivity, reproducibility, and throughput suitable for large-scale phosphoproteomic studies. The method reduces sample-to-sample variability, facilitates parallel processing of up to 96 samples, and enables reliable quantification of phosphorylation sites in diverse biological samples.

Budoucí trendy a možnosti využití

• Integration with multi-enzyme workflows and alternative metal-chelate phases (e.g., TiO₂, Zr(IV)).
• Development of combinatorial enrichment strategies to capture mono- and multiphosphorylated peptides sequentially.
• Application to low-input or clinical specimens, single-cell proteomics, and high-throughput drug screening.
• Further miniaturization and real-time monitoring of binding/elution kinetics on automated platforms.

Závěr

The Agilent AssayMAP Bravo combined with high-capacity Fe(III)-NTA cartridges provides a robust, automated workflow for phosphopeptide enrichment. Through systematic optimization of chemistry and loading parameters, the method achieves high specificity, extensive coverage, and outstanding reproducibility, meeting the demands of modern phosphoproteomic research.

Reference

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