Human Breast Cancer Cell Line Phosphoproteome Revealed by an Automated and Highly Selective Enrichment Workflow

Význam tématu

Analyses of phosphoproteomes provide insights into cell signaling pathways and disease mechanisms. Reliable enrichment of low-abundance phosphopeptides is essential for reproducible mass spectrometry–based phosphoproteomics in research and quality control.

Cíle a přehled studie

This study evaluated the performance and reproducibility of an automated phosphopeptide enrichment workflow using Agilent AssayMAP Bravo with high-capacity Fe(III)-NTA cartridges. By varying sample/resin ratios (R1–R4) and spiking with light and heavy isotopic phosphopeptide mixes, the aim was to assess enrichment yield, selectivity, and identification depth.

Použitá metodika a instrumentace

• Sample preparation: MCF7 cell line tryptic digests spiked with 20 light phosphopeptide standards.
• Enrichment: Agilent AssayMAP Bravo v2.0 with Fe(III)-NTA cartridges, four distinct sample-to-resin ratios.
• Discovery proteomics: Agilent 1290 Infinity II UHPLC with Nanodapter to nanoflow, coupled to 6550 iFunnel Q-TOF MS; data-dependent acquisition; identification by Spectrum Mill at 1.5% FDR.
• Quantitation: Agilent 6495B triple quadrupole LC/MS with MRM transitions optimized via Skyline; heavy standards spiked post-enrichment for yield calculation.

Hlavní výsledky a diskuse

• Enrichment selectivity improved from ~1.5% before to over 90% after across all ratios.
• Distinct phosphopeptide identifications ranged from ~1,513 (R1) to ~1,188 (R4), increasing with higher sample loads though not strictly proportional.
• Average enrichment yield of the 16 detected standards was ~63% with <5% RSD across ratios, demonstrating high reproducibility.
• Individual peptide yields varied (4–100%), reflecting sequence-dependent binding efficiencies.

Přínosy a praktické využití metody

The automated workflow reduces manual variability, enhances throughput, and delivers consistent high selectivity, supporting both sensitive discovery and targeted quantitative phosphoproteomics. Integration with nanoflow LC/MS and Skyline streamlines method development and data processing.

Budoucí trendy a možnosti využití

• Miniaturization and higher-capacity cartridges for lower sample inputs.
• Automated bioinformatics pipelines for real-time data analysis.
• Multiplexed quantitation with novel isotopic labeling strategies.
• Applications in clinical, single-cell, and spatial phosphoproteomics.

Závěr

The Agilent AssayMAP Bravo automated enrichment workflow with Fe(III)-NTA cartridges offers robust, reproducible phosphopeptide enrichment from complex cell digests across variable sample loads. Coupled with nanoflow LC–MS and automated MRM quantitation in Skyline, it provides a versatile platform for comprehensive phosphoproteomic studies.

Reference

• Wu S, Wu L. Agilent AssayMAP Bravo enables reproducible automated phosphopeptide enrichment using Fe(III)-NTA cartridges. Agilent Technologies Application Note 5991-6073EN (2018).
• Phosphomix peptide standards. Sigma-Aldrich, 2018.
• Agilent Nanodapter for discovery proteomics using nanoflow LC/MS. Application Note 5991-8174EN (2017).
• Agilent 6495 Triple Quadrupole LC/MS: Peptide quantitation performance. Application Note 5991-6898EN (2017).
• Agilent triple quadrupole LC/MS peptide quantitation with Skyline. Application Note 5990-9887EN (2016).