Sequential enrichment using metal oxide affinity chromatography (SMOAC) to enhance phosphoproteome coverage for quantitative proteomic analysis

Importance of Topic

Phosphorylation is a crucial post-translational modification that governs protein function, localization, and interactions in cellular signaling networks. Its low stoichiometry in complex mixtures necessitates selective enrichment strategies to enable comprehensive mass spectrometry–based phosphoproteome analysis.

Objectives and Study Overview

This study compared two sequential enrichment workflows—SIMAC (Fe-NTA followed by TiO2) and the novel SMOAC (TiO2 followed by Fe-NTA)—using Thermo Scientific High-Select kits. It further integrated high-pH reversed-phase fractionation and tandem mass tag (TMT) multiplexing to achieve both deep coverage and robust quantitative phosphoproteomic profiling.

Methodology and Instrumentation

• Cell culture: HeLa suspension and adherent cells treated with nocodazole, serum starvation, growth factors (EGF, IGF-1, PDGF), TPA, or vehicle.
• Protein digestion: Denaturation in urea, reduction with DTT, alkylation with iodoacetamide, sequential Lys-C and trypsin proteolysis.
• Enrichment: High-Select TiO2 and Fe-NTA Phosphopeptide Enrichment Kits applied as SIMAC or SMOAC workflows.
• Fractionation: High-pH reversed-phase spin columns to reduce sample complexity and improve peptide separation.
• Quantitation: TMT10plex and TMT11-131C labeling for up to eleven-plex relative quantification.
• LC-MS/MS: Orbitrap Fusion Tribrid mass spectrometer, full scan at 120 000 resolution, HCD MS2 at 50 000 resolution.
• Data analysis: Proteome Discoverer 2.2 with SEQUEST HT, precursor tolerance 10 ppm, fragment tolerance 0.02 Da, phosphoRS site localization, 1% FDR, TMT reporter ion quantification.

Main Results and Discussion

SMOAC outperformed SIMAC by yielding ~13 300 additional unique phosphopeptides in the second enrichment step, compared with ~2 900 for SIMAC. Sequential TiO2→Fe-NTA (SMOAC) provided a 21% increase in unique phosphopeptides versus parallel enrichment. Incorporation of high-pH fractionation expanded identifications to over 32 500 unique phosphopeptides. In an eleven-plex TMT experiment, nearly 24 000 phosphorylation sites were confidently quantified, revealing dynamic changes in key regulators such as CDC25 family members and uncovering novel modification sites.

Benefits and Practical Applications

• Enhanced phosphoproteome depth and specificity.
• Reduced sample requirements via sequential enrichment.
• Seamless integration with TMT multiplexing for high-throughput quantitation.
• Improved discovery of novel phosphorylation sites and signaling events.

Future Trends and Potential Applications

Future developments may include coupling SMOAC with data-independent acquisition (DIA) workflows, next-generation mass spectrometers for faster acquisition, machine learning–driven enrichment optimization, and clinical phosphoproteomics for biomarker discovery and personalized medicine applications.

Conclusion

The SMOAC workflow combining TiO2 and Fe-NTA sequential enrichment, high-pH fractionation, and TMT multiplexed quantitation on an Orbitrap platform achieves substantially deeper and more comprehensive phosphoproteome coverage than existing methods, enabling robust analysis of phosphorylation dynamics across diverse biological conditions.

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