Quantitative analysis of signaling pathways using TMT 11plex reagents and comprehensive phosphopeptide enrichment strategies

Importance of the topic

Understanding dynamic changes in protein phosphorylation is essential for elucidating cellular signaling and disease mechanisms. However, the low abundance and transient nature of many phosphorylation events pose analytical challenges. Advances in multiplexed quantitative phosphoproteomics can transform pathway analysis by enabling simultaneous profiling across multiple conditions.

Objectives and Study Overview

This study aimed to integrate an 11-plex Tandem Mass Tag (TMT) labeling strategy with a Sequential Metal Oxide Affinity Chromatography (SMOAC) workflow and high-pH reversed-phase fractionation to achieve comprehensive phosphoproteome quantitation in HeLa cells under ten distinct stimulation conditions.

Methodology and Instrumentation

• Cell Culture and Treatment: HeLa cells were subjected to ten conditions including growth factors (EGF, IGF-1, PDGF-bb), TPA, nocodazole, serum stimulation, and serum starvation.
• Protein Processing: Lysates were reduced, alkylated, digested, and labeled with TMT10plex reagents plus a novel TMT11-131C for multiplexing 11 samples.
• Phosphopeptide Enrichment (SMOAC): Initial enrichment with TiO2 followed by pooling of flow-through and washes and secondary enrichment with Fe-NTA.
• Fractionation: Combined eluates were fractionated using high-pH reversed-phase chromatography into eight fractions.
• LC-MS Analysis: Peptides were separated on a 50 cm EASY-Spray C18 column and analyzed on an Orbitrap Fusion instrument using high-resolution HCD MS2.
• Data Analysis: Proteome Discoverer 2.2 with Byonic/Sequest HT search engines and PhosphoRS for site localization; custom TMT11plex quantification with 1% FDR.

Main Results and Discussion

• Identification: Over 33 000 phosphopeptides identified, with ~24 000 quantified and localized at >90% confidence.
• Workflow Performance: SMOAC coupled with high-pH fractionation doubled phosphopeptide yield compared to TiO2 alone, while maintaining high specificity.
• Multiplex Quantitation: Enabled simultaneous analysis of phosphorylation changes across ten cellular treatments plus a pooled reference in under 48 hours of instrument time.
• Pathway Insights: Differential phosphorylation profiles mapped to key signaling cascades (e.g., mTOR, MAPK, insulin, FoxO, AMPK) via KEGG analysis.
• Validation: Site-specific changes in RAF1 phosphorylation (pSer259, pSer289, pSer296) were confirmed by orthogonal methods (western blot and IP-MS), showing strong correlation with MS data.

Benefits and Practical Applications

• Enhanced Depth: High coverage phosphoproteome mapping from low-abundance sites.
• Throughput: Eleven-sample multiplexing reduces instrument time and increases experimental scope.
• Quantitative Accuracy: Isobaric labeling with TMT11plex ensures consistent relative quantification across conditions.
• Versatility: Applicable to diverse cell types and treatments for signaling network analysis in research and drug development.

Used Instrumentation

• Thermo Scientific Orbitrap Fusion Tribrid Mass Spectrometer
• Thermo Scientific EASY-Spray C18 LC Column (50 cm, 2 µm)
• Thermo Scientific Pierce TiO2 and Fe-NTA Phosphopeptide Enrichment Kits
• Thermo Scientific Pierce High-pH Reversed-Phase Peptide Fractionation Kit
• Thermo Scientific Proteome Discoverer 2.2 Software

Future Trends and Opportunities

• Expansion to higher plexing reagents for broader comparative studies.
• Integration with real-time metrics and machine learning for dynamic pathway modeling.
• Application to clinical samples for biomarker discovery and personalized medicine.
• Combination with complementary enrichment strategies (e.g., antibody-based) to capture other PTMs.

Conclusion

The integration of an innovative 11-plex TMT labeling strategy with SMOAC and high-pH fractionation significantly improves phosphoproteome coverage and quantitation throughput. This workflow enables robust comparative analysis of signaling pathways, providing a powerful tool for basic and translational research in cell biology and drug discovery.

References

• Lombardi B et al. EuPA Open Proteomics 2015;6:10–15.
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• Thermo Fisher Scientific Application Note #566, 2012.
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• Käll L et al. Nat Methods. 2007;4:923–925.