Phospho-EIF4EBP1 (Thr36) Antibody

What is the biological function of 4E-BP1 and why is its phosphorylation at Thr36 significant?

4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) is a key translation repressor protein that directly interacts with eIF4E, a limiting component of the multisubunit complex that recruits 40S ribosomal subunits to the 5' end of mRNAs. In its non-phosphorylated form, 4E-BP1 binds to eIF4E and prevents its interaction with eIF4G, thereby inhibiting complex assembly and repressing cap-dependent translation .

Phosphorylation of 4E-BP1 at Thr36 (Thr37 in humans) is particularly significant as it serves as a priming event in the sequential phosphorylation process. This initial phosphorylation event, often followed by phosphorylation at other sites (Thr46, Thr70, and Ser65), disrupts the inhibitory interaction with eIF4E, allowing cap-dependent translation to proceed . The phosphorylation status of 4E-BP1 at Thr36/37 is therefore a critical regulatory mechanism for protein synthesis and cell growth, with important implications for various physiological and pathological processes.

How does phosphorylation at Thr36 relate to other phosphorylation sites on 4E-BP1?

The phosphorylation of 4E-BP1 follows a hierarchical pattern where multiple sites are involved. Seven phosphorylation sites have been identified in human 4E-BP1: Thr37, Thr46, Ser65, Thr70, Ser83, Ser101, and Ser112 . The phosphorylation sequence typically begins with Thr37 and Thr46 (equivalent to Thr36 and Thr45 in mouse and rat), which serve as priming events, followed by Thr70 phosphorylation and finally Ser65 phosphorylation .

This hierarchical phosphorylation is functionally significant because:

• Phosphorylation at Thr37/46 (Thr36/45) alone is insufficient to completely disrupt 4E-BP1 binding to eIF4E

• Complete dissociation requires additional phosphorylation at Ser65 and Thr70

• The multi-site phosphorylation creates a higher threshold for susceptibility to degradation, potentially contributing to protein stabilization

Understanding the relationship between these phosphorylation sites is crucial for interpreting experimental results when using phospho-specific antibodies targeting individual sites.

What are the validated applications for Phospho-EIF4EBP1 (Thr36) antibodies and how should they be optimized?

Phospho-EIF4EBP1 (Thr36) antibodies have been validated for multiple experimental applications. Based on current research and manufacturer specifications, these include:

For optimization:

• When using for flow cytometry, follow Protocol A: Two-step protocol for intracellular (cytoplasmic) proteins or Protocol C: Two-step protocol with Fixation/Methanol for the greatest discrimination of phospho-specific signaling between unstimulated and stimulated samples

• For Western blotting, positive control samples such as EGF-treated (200 ng/ml, 30min) MDA-MB-435 cells can help validate specificity

• For immunohistochemistry, peptide competition assays are recommended to confirm specificity, as demonstrated with human breast carcinoma tissue samples

How can I properly validate the specificity of a Phospho-EIF4EBP1 (Thr36) antibody?

Validating antibody specificity is critical for reliable research. For Phospho-EIF4EBP1 (Thr36) antibodies, consider the following methodological approaches:

• Peptide competition assays: Compare staining with and without pre-incubation with the immunizing phosphopeptide. A significant reduction in signal when using the blocking peptide confirms specificity, as demonstrated in immunohistochemical analysis of human breast carcinoma tissue

• Phosphatase treatment: Treat half of your sample with lambda phosphatase to remove phosphate groups and compare with untreated samples. Loss of signal in treated samples confirms phospho-specificity

• Kinase inhibitor experiments: Treat cells with specific inhibitors of known upstream kinases that phosphorylate 4E-BP1 at Thr36, such as PI3 kinase inhibitors LY294002 and wortmannin, which inhibit mTOR-mediated phosphorylation

• Genetic models: Use 4E-BP1 knockout cells or cells expressing phospho-deficient mutants (T36A) as negative controls. Western blotting analysis using 4E-BP1 and 4E-BP2 singly deficient lymphocytes has been used to confirm band specificity

• Stimulation experiments: Compare unstimulated cells with those treated with known activators of the mTOR pathway (e.g., insulin, EGF). As demonstrated in research, EGF treatment (200 ng/ml, 30min) of MDA-MB-435 cells significantly increases Thr36 phosphorylation

These validation approaches should be documented and included in publications to ensure reproducibility and reliability of results.

How can I differentiate between phosphorylation of 4E-BP1 at Thr36 versus Thr37/46 in experimental analysis?

Differentiating between closely spaced phosphorylation sites presents a technical challenge due to antibody cross-reactivity and sequence similarity. To address this specific issue:

• Understand antibody specificity: Many commercial antibodies detect both Thr36 and Thr37/46 phosphorylation. For example, the V3NTY24 monoclonal antibody recognizes both human and mouse 4E-BP1 when phosphorylated at threonine 37 and/or threonine 46 . Review the exact epitope used for antibody generation in product datasheets.

• Use site-specific mutants: Generate expression constructs with single-site mutations (T36A, T37A, or T46A) to distinguish the contribution of each site to the observed signal.

• Mass spectrometry analysis: For definitive site identification, employ phospho-site mapping by mass spectrometry, which can precisely identify the phosphorylated residues.

• Peptide arrays: Use peptide arrays containing the various phospho-sites to test antibody specificity and cross-reactivity.

• Sequential immunoprecipitation: First immunoprecipitate with one phospho-specific antibody, then probe the supernatant with another to separate populations of differently phosphorylated 4E-BP1.

It's important to note that in rodents, the phosphorylation site numbering is one lower than in humans (Thr36 in rodents corresponds to Thr37 in humans) , which should be considered when interpreting cross-species studies.

What are common pitfalls when interpreting Phospho-EIF4EBP1 (Thr36) antibody results and how can they be avoided?

Several challenges can affect the interpretation of Phospho-EIF4EBP1 (Thr36) antibody results:

• Species-specific numbering confusion: Thr36 in rodents corresponds to Thr37 in humans . Always verify which numbering system your antibody documentation uses and report accordingly.

• Hierarchical phosphorylation effects: Since 4E-BP1 phosphorylation follows a sequential pattern, changes in upstream phosphorylation events may affect Thr36 phosphorylation indirectly. Include detection of other phosphorylation sites (Thr46, Ser65, Thr70) when possible.

• Gel migration pattern complexity: Phosphorylation affects the SDS-PAGE migration of 4E-BP1, with multiple bands representing different phosphorylation states. Extensive analysis has shown that Ser65 phosphorylation contributes substantially to the SDS-PAGE migration pattern . Use phospho-site specific antibodies in combination with total 4E-BP1 antibodies to interpret band shifts correctly.

• 4E-BP1 vs 4E-BP2 confusion: Both proteins can be detected in the same samples with different phosphorylation sensitivities. In lymphocytes, for example, 4E-BP2 shows greater rapamycin sensitivity than 4E-BP1 . Use isoform-specific knockout controls to validate band identity.

• Kinase inhibitor off-target effects: Inhibitors used to modulate 4E-BP1 phosphorylation may have off-target effects. Include multiple inhibitors with different mechanisms and appropriate controls.

To avoid these pitfalls:

• Always include both phospho-specific and total protein antibodies

• Use multiple antibodies targeting different epitopes when available

• Include appropriate positive and negative controls

• Consider genetic approaches (knockouts, site-specific mutants) for validation

• Report the exact antibody clone and catalog number in publications

How can Phospho-EIF4EBP1 (Thr36) antibodies be used to investigate mTOR-independent phosphorylation pathways?

While mTOR is traditionally associated with 4E-BP1 phosphorylation, recent research has identified several mTOR-independent kinases that can phosphorylate 4E-BP1 at Thr36/37. Investigating these alternative pathways requires strategic experimental approaches:

• Rapamycin and mTOR kinase inhibitor comparisons: Utilize both rapamycin (which incompletely inhibits mTOR) and ATP-competitive mTOR kinase inhibitors like MLN0128 (which more completely block mTOR). Differences in 4E-BP1 phosphorylation between these treatments may reveal mTOR-independent mechanisms .

• Stress condition analysis: Examine 4E-BP1 phosphorylation under various stress conditions where mTOR is inhibited but alternative pathways are activated:

  • UVB irradiation activates p38MAPK and MSK1-mediated phosphorylation

  • Ionizing radiation triggers ATM-dependent ERK phosphorylation of 4E-BP1

  • Hypoxia shifts from cap-dependent to cap-independent translation

• Alternative kinase targeting: Employ specific inhibitors for non-mTOR kinases reported to phosphorylate 4E-BP1:

  • p38MAPK inhibitors for UV-activated pathways

  • PIM kinase inhibitors (PIM2 phosphorylates Ser65)

  • CDK1 inhibitors (phosphorylates Thr70)

  • LRRK2 inhibitors (phosphorylates Thr37/46)

• Genetic approaches: Use CRISPR/Cas9 or siRNA to knockdown specific kinases and assess their contribution to 4E-BP1 phosphorylation at Thr36/37.

• Structural variations: Investigate how amino acid variations around phosphorylation sites affect kinase specificity. For instance, research has shown that mutation of Gly31 to glutamine or histidine rendered Thr37/46 in 4E-BP1 more rapamycin-sensitive .

These approaches can reveal the complex regulation of 4E-BP1 phosphorylation beyond the canonical mTOR pathway, with important implications for drug resistance mechanisms in cancer and other diseases.

What is the significance of differential 4E-BP1 versus 4E-BP2 phosphorylation patterns in various cell types?

The 4E-BP family includes 4E-BP1, 4E-BP2, and 4E-BP3, with distinct expression patterns and phosphorylation sensitivities across cell types. This differential regulation has significant biological implications:

• Lymphocyte-specific regulation: Lymphocytes have increased amounts of 4E-BP2, which shows greater rapamycin sensitivity than 4E-BP1. Research demonstrates that in activated B cells, rapamycin reduces phosphorylation of 4E-BP2 but not 4E-BP1 on Thr36/45, while the TOR-KI compound MLN0128 suppresses phosphorylation of both . This suggests that the 4E-BP–eIF4E axis is uniquely rapamycin-sensitive in lymphocytes, promoting clonal expansion of these cells.

• Sequence determinants of phosphorylation sensitivity: Analysis of sequence alignments revealed a conserved glycine at position 31 of mouse 4E-BP1, a position occupied by a polar amino acid residue (glutamine or histidine) in 4E-BP2. Experimental mutation of Gly31 to glutamine or histidine rendered Thr37/46 in 4E-BP1 more rapamycin-sensitive , highlighting how subtle sequence variations determine phosphorylation dynamics.

• Tissue-specific functions: Different cell types exhibit varying ratios of 4E-BP1 and 4E-BP2, which may influence their response to stress conditions, growth factors, and therapeutic interventions. For studying these differences, researchers should:

  • Use isoform-specific antibodies

  • Employ genetic models with selective isoform knockouts

  • Compare primary cells from different tissues within the same organism

  • Analyze phosphorylation patterns in response to various stimuli and inhibitors

These differences have direct implications for therapeutic approaches targeting the mTOR pathway, suggesting that tissue-specific responses may depend on the predominant 4E-BP isoform and its particular phosphorylation sensitivity.

How can Phospho-EIF4EBP1 (Thr36) status be effectively used as a biomarker in cancer research?

The phosphorylation status of 4E-BP1 at Thr36/37 has emerging value as a biomarker in cancer research, with several methodological considerations for optimal implementation:

• Tissue preservation and processing: Phosphorylation states can rapidly change during tissue collection and processing. Implement standardized protocols including:

  • Rapid tissue fixation (within 15-30 minutes of collection)

  • Use of phosphatase inhibitors in all buffers

  • Consistent fixation times for comparative studies

  • Documentation of ischemia time

• Quantification approaches: For immunohistochemistry applications, utilize:

  • Digital image analysis rather than manual scoring

  • Multiplex staining including total 4E-BP1 and other phospho-sites

  • Ratio of phosphorylated to total protein rather than absolute values

  • Internal controls on each slide for normalization

• Relevant controls and cutoffs: Establish:

  • Appropriate positive controls (e.g., EGF-treated MDA-MB-435 cells )

  • Negative controls including peptide competition assays

  • Clinically meaningful cutoff values through ROC curve analysis

  • Correlation with other established biomarkers

• Integration with other markers: Phospho-4E-BP1 (Thr36) should be evaluated in the context of:

  • Upstream pathway activation (PI3K/AKT/mTOR)

  • Downstream effectors of protein synthesis

  • Other phosphorylation sites on 4E-BP1

  • Indicators of treatment response

Research has shown that hyperphosphorylation and overexpression of 4E-BP1 occurs simultaneously in human cancers , making it important to assess both parameters. Additionally, understanding the relationship between 4E-BP1 phosphorylation status and treatment response can help guide personalized therapy approaches, particularly for mTOR inhibitors.

What methodological considerations are important when studying Phospho-EIF4EBP1 (Thr36) in the context of drug resistance mechanisms?

Investigating the role of Phospho-EIF4EBP1 (Thr36) in drug resistance, particularly to mTOR inhibitors, requires careful experimental design:

• Time-course analyses: Short-term versus long-term drug exposure can reveal adaptive mechanisms:

  • Acute responses (minutes to hours) often reflect direct signaling effects

  • Chronic responses (days to weeks) may involve compensatory mechanisms

  • Pulsatile treatment schedules can distinguish between transient and sustained effects

• Multiple inhibitor approach: Compare:

  • Rapalogs (rapamycin, everolimus) which show incomplete inhibition of 4E-BP1 phosphorylation

  • ATP-competitive mTOR kinase inhibitors (MLN0128) with more complete inhibition

  • Dual PI3K/mTOR inhibitors to address upstream compensation

  • Inhibitors of alternative kinases identified to phosphorylate 4E-BP1 (p38MAPK, PIM2, CDK1, LRRK2)

• Resistance model development:

  • Generate resistant cell lines through chronic drug exposure

  • Compare matched sensitive/resistant pairs derived from the same parental line

  • Use patient-derived xenografts from treatment-naïve and post-progression samples

  • Employ genetic engineering to modulate specific resistance mechanisms

• Integrated pathway analysis:

  • Assess multiple phosphorylation sites on 4E-BP1 (Thr36/37, Thr46, Ser65, Thr70)

  • Examine alternative translation mechanisms (cap-independent translation)

  • Investigate potential mTOR-independent kinases activating 4E-BP1

  • Evaluate 4E-BP1:eIF4E binding status using cap-binding assays

Research has shown that phosphorylation of 4E-BP1 can become resistant to rapamycin through induction of PIM kinases , and PIM2 can directly phosphorylate 4E-BP1 at Ser65 . Additionally, multi-site phosphorylation may play a role in protein stabilization and overexpression , which could contribute to drug resistance mechanisms.

By implementing these methodological approaches, researchers can better understand the complex role of 4E-BP1 phosphorylation in treatment resistance and develop strategies to overcome it.

How should Phospho-EIF4EBP1 (Thr36) antibodies be optimized for flow cytometry applications?

Flow cytometry with phospho-specific antibodies requires careful optimization to achieve reliable results when detecting Phospho-EIF4EBP1 (Thr36):

• Sample preparation and fixation:

  • For optimal results, use Protocol A (two-step protocol for intracellular cytoplasmic proteins) or Protocol C (two-step protocol with Fixation/Methanol)

  • The methanol-based protocol allows for the greatest discrimination of phospho-specific signaling between unstimulated and stimulated samples

  • Fix cells within minutes of collection to preserve phosphorylation status

  • Include phosphatase inhibitors in all buffers prior to fixation

• Antibody selection and titration:

  • The V3NTY24 monoclonal antibody has been pre-titrated and tested for flow cytometry

  • Use at 5 μL (0.06 μg) per test (defined as the amount of antibody that will stain a cell sample in a final volume of 100 μL)

  • Cell numbers can range from 10^5 to 10^8 cells/test but should be determined empirically

  • Available conjugates include PE and eFluor 660, allowing flexibility in panel design

• Controls and validation:

  • Include unstimulated versus stimulated samples (e.g., with insulin or EGF)

  • Use inhibitor-treated cells (PI3K inhibitors LY294002 or wortmannin) as negative controls

  • Include isotype controls with matching fluorophores

  • When possible, include genetic controls (4E-BP1 knockout cells)

• Data analysis considerations:

  • Analyze phospho-signal as median fluorescence intensity rather than percent positive

  • Calculate phosphorylation index as the ratio of stimulated to unstimulated signals

  • Consider co-staining for total 4E-BP1 to normalize phospho-signal

  • For heterogeneous samples, use lineage markers to identify cell subpopulations

These technical recommendations will help ensure robust and reliable detection of Phospho-EIF4EBP1 (Thr36) in flow cytometry applications, particularly important for analyzing primary cells and heterogeneous samples where western blotting might not reveal cell-specific differences.

What are the optimal approaches for multiplexed detection of different 4E-BP1 phosphorylation sites?

Multiplexed detection of multiple 4E-BP1 phosphorylation sites provides comprehensive insight into its regulation. Several technical approaches can be employed:

• Multiplexed western blotting:

  • Sequential probing: Strip and reprobe membranes with different phospho-specific antibodies

  • Multiple molecular weight regions: Take advantage of the different migration patterns of phosphorylated 4E-BP1 forms

  • Fluorescent detection: Use spectrally distinct secondary antibodies to simultaneously detect different phospho-sites

  • Semi-quantitative analysis: Compare phosphorylation at different sites using densitometry normalized to total 4E-BP1

• Multiplexed immunohistochemistry/immunofluorescence:

  • Sequential staining protocols with antibody stripping between rounds

  • Tyramide signal amplification to allow multiple primary antibodies from the same species

  • Spectral unmixing for closely overlapping fluorophores

  • Multi-spectral imaging systems for quantitative analysis

• Bead-based multiplex assays:

  • Custom multiplex bead arrays with different phospho-4E-BP1 antibodies

  • Simultaneous detection of total 4E-BP1 and multiple phospho-sites

  • Inclusion of upstream pathway components (mTOR, AKT) and downstream targets

• Mass cytometry (CyTOF):

  • Metal-tagged antibodies allow simultaneous detection of 30+ parameters

  • Include antibodies against total 4E-BP1, multiple phospho-sites, and relevant pathway components

  • Single-cell resolution reveals heterogeneity in 4E-BP1 phosphorylation

• Phospho-proteomic approaches:

  • Targeted mass spectrometry for absolute quantification of all phosphorylation sites

  • Phospho-peptide enrichment prior to analysis

  • SILAC or TMT labeling for comparative studies

  • Data correlation with antibody-based detection methods

When implementing these approaches, ensure antibody compatibility (primary antibody host species, isotypes, epitope interference), include appropriate controls for each phospho-site, and validate the specificity of the multiplexed assay against single-site detection methods to confirm no cross-reactivity or interference occurs.

How might new technologies enhance our understanding of 4E-BP1 phosphorylation dynamics?

Emerging technologies offer exciting opportunities to deepen our understanding of 4E-BP1 phosphorylation dynamics:

• Live-cell imaging of phosphorylation events:

  • FRET-based biosensors for real-time monitoring of 4E-BP1 phosphorylation

  • Split luciferase complementation assays to measure 4E-BP1-eIF4E interaction dynamics

  • Optogenetic control of mTOR activity to precisely manipulate 4E-BP1 phosphorylation

• Single-cell analysis approaches:

  • Single-cell phospho-proteomics to reveal cell-to-cell heterogeneity

  • Mass cytometry with imaging capabilities (IMC) to preserve spatial information

  • Single-cell western blotting for simultaneous detection of multiple proteins

• Spatial biology techniques:

  • Highly multiplexed imaging (CODEX, Hyperion) to visualize 4E-BP1 phosphorylation in tissue context

  • Spatial transcriptomics correlated with protein phosphorylation

  • 3D tissue clearing with whole-organ phospho-protein imaging

• Structural biology advances:

  • Cryo-EM structures of 4E-BP1 in different phosphorylation states

  • Hydrogen-deuterium exchange mass spectrometry to detect conformational changes

  • NMR studies of phosphorylation-induced structural transitions

• Computational approaches:

  • Machine learning algorithms to predict phosphorylation patterns from multiple inputs

  • Mathematical modeling of the hierarchical phosphorylation cascade

  • Systems biology integration of phosphorylation data with other -omics datasets

These technologies will help address key questions: How does the temporal sequence of multi-site phosphorylation unfold at the single-molecule level? How do different cell types and microenvironments influence 4E-BP1 phosphorylation patterns? How does the 3D structure of 4E-BP1 change with each sequential phosphorylation event?

By embracing these innovative approaches, researchers can build a more comprehensive understanding of 4E-BP1 regulation that spans from molecular interactions to cellular heterogeneity and tissue-level effects.

What are the emerging roles of Phospho-EIF4EBP1 (Thr36) beyond translation regulation?

Recent research suggests that phosphorylated 4E-BP1 may have functions beyond its canonical role in translation regulation:

• Potential nuclear functions:

  • While traditionally viewed as cytoplasmic, recent evidence suggests that 4E-BP1 may have nuclear functions

  • Phosphorylated 4E-BP1 might interact with nuclear proteins involved in transcription or mRNA processing

  • Research methodologies should include nuclear/cytoplasmic fractionation and co-immunoprecipitation studies to identify novel interaction partners

• Stress granule dynamics:

  • Under stress conditions, mRNA translation is regulated through stress granule formation

  • Phosphorylation status of 4E-BP1 may influence stress granule assembly and disassembly

  • Fluorescence recovery after photobleaching (FRAP) and live-cell imaging approaches can investigate this relationship

• Liquid-liquid phase separation:

  • Many translation factors participate in biomolecular condensate formation

  • Phosphorylation of 4E-BP1 likely affects its partitioning between different cellular compartments

  • In vitro reconstitution systems and advanced microscopy can explore these properties

• Alternative translation mechanisms:

  • Research has revealed that cytoplasmic overexpressed 4E-BP1 orchestrates a hypoxia-activated switch from cap-dependent to cap-independent mRNA translation

  • This promotes increased tumor angiogenesis and growth through selective mRNA translation of factors like VEGF-A, HIF1α, and Bcl2

  • Ribosome profiling and polysome analysis can reveal the complete spectrum of mRNAs affected

• Metabolic regulation:

  • Emerging evidence suggests crosstalk between 4E-BP1 phosphorylation and cellular metabolic pathways

  • Phosphorylated 4E-BP1 may participate in feedback loops with nutrient sensing mechanisms

  • Metabolomic approaches integrated with phospho-protein analysis can illuminate these connections

The observation that 70% or more of cellular 4E-BP1 is not bound to eIF4E raises important questions about its additional functions . Exploring these non-canonical roles requires innovative experimental approaches combining traditional biochemical methods with emerging technologies that can capture the dynamic, contextual nature of protein function.

What specialized techniques are recommended for studying Phospho-EIF4EBP1 (Thr36) in primary cells and tissues?

Working with primary cells and tissues presents unique challenges for phospho-protein analysis that require specialized approaches:

• Rapid sample processing protocols:

  • Preserve phosphorylation status by minimizing time between tissue collection and fixation/lysis

  • Use vacuum-assisted tissue collection systems when possible

  • Employ stabilization reagents specifically designed for phospho-proteins

  • Document cold ischemia time for all samples

• Laser capture microdissection (LCM):

  • Isolate specific cell types from heterogeneous tissues

  • Combine with phospho-specific antibodies for region-specific analysis

  • Implement modified protein extraction protocols optimized for small sample input

  • Consider subsequent analysis by highly sensitive nano-immunoassays

• Ex vivo tissue slice cultures:

  • Maintain tissue architecture while allowing experimental manipulation

  • Treat with kinase inhibitors/activators directly in culture

  • Use phospho-specific immunofluorescence for spatial resolution

  • Combine with metabolic labeling to track newly synthesized proteins

• Primary cell isolation considerations:

  • Modify isolation procedures to maintain phosphorylation status

  • Include phosphatase inhibitors throughout isolation process

  • Implement gentle cell separation techniques (magnetic sorting over FACS when possible)

  • Compare freshly isolated cells with those subjected to short-term culture

• Validation in multiple species:

  • Account for species differences in phosphorylation site numbering (Thr36 in rodents = Thr37 in humans)

  • Test antibody cross-reactivity empirically despite predicted homology

  • Consider species-specific sequence variations that may affect kinase recognition

  • Use of site-specific mutants expressed in cells from relevant species

These methodological approaches will help ensure that phosphorylation data obtained from primary cells and tissues accurately reflects the in vivo state, providing more translatable insights into 4E-BP1 biology across different physiological and pathological contexts.

How can isotope-coded affinity tag (ICAT) and other quantitative proteomic approaches be applied to study 4E-BP1 phosphorylation networks?

Quantitative proteomics offers powerful approaches to comprehensively analyze 4E-BP1 phosphorylation networks:

• Phospho-specific enrichment strategies:

  • Titanium dioxide (TiO2) or immobilized metal affinity chromatography (IMAC) for global phospho-peptide enrichment

  • Phospho-tyrosine antibody enrichment followed by serial enrichment for phospho-serine/threonine

  • Combinatorial use of multiple enrichment methods to maximize coverage

• Isotope labeling approaches:

  • SILAC (Stable Isotope Labeling with Amino acids in Cell culture) for cell line comparisons

  • TMT (Tandem Mass Tag) or iTRAQ (isobaric Tags for Relative and Absolute Quantification) for multiplexing up to 16 conditions

  • ICAT (Isotope-Coded Affinity Tags) specifically targeting cysteine-containing peptides

  • Label-free quantification for tissue samples or primary cells

• Targeted mass spectrometry:

  • Multiple Reaction Monitoring (MRM) or Parallel Reaction Monitoring (PRM) for specific phospho-sites

  • Heavy-labeled synthetic phospho-peptide standards for absolute quantification

  • Sequential Windowed Acquisition of All Theoretical fragment ion Mass Spectra (SWATH-MS) for comprehensive detection

• Kinase-substrate relationship mapping:

  • Kinase inhibitor panels combined with phospho-proteomics

  • ATP analog approaches for direct kinase substrate identification

  • Correlation analysis of kinase activity and substrate phosphorylation

  • Computational integration of phosphorylation motifs with quantitative data

• Integrated network analysis:

  • Map 4E-BP1 phosphorylation sites in context of broader signaling networks

  • Identify co-regulated phosphorylation events during drug treatment

  • Reveal compensatory phosphorylation events in resistance mechanisms

  • Integrate phospho-proteomics with transcriptomics and metabolomics