Protocol for Phosphoproteomes Analysis by Selective Labelling and Mass Spectrometric Techniques

Why Choose Selective Labelling and Advanced Mass Spectrometric Techniques?

Unveiling the Complexity of Phosphoproteomics

The elucidation of phosphoproteomics necessitates methodologies capable of capturing the dynamic nature and complexity of protein phosphorylation events. Traditional approaches focusing solely on protein abundance fail to encapsulate the nuanced regulatory roles exerted by PTMs. By employing selective labeling strategies, coupled with advanced mass spectrometry (MS) techniques, researchers can delve deep into the phosphoproteomic landscape, deciphering intricate signaling networks and molecular interactions.

Enhancing Sensitivity and Specificity

Selective labeling techniques enable the enrichment of phosphorylated peptides amidst complex proteomic mixtures, enhancing the sensitivity and specificity of phosphoproteome analysis. Advanced MS instruments, such as MALDI-TOF and quadrupole/ion trap hybrids, offer unparalleled resolution and accuracy, facilitating the identification and quantification of phosphorylation events with high precision.

Unraveling Signaling Dynamics

The dynamic nature of protein phosphorylation necessitates methodologies capable of capturing transient modifications and signaling dynamics. Selective labeling coupled with time-resolved MS analysis provides insights into the temporal regulation of phosphorylation events, unveiling the kinetics and kinetics of cellular signaling pathways.

Material for Phosphoproteomes Analysis by Selective Labelling and Advanced Mass Spectrometric Techniques

Protein Digestion

1. o-Cascin Water Solution: 1 ug/ul concentration, stored at -20°C.

2. Buffer Solution: 0.1 M ammonium bicarbonate (AMBIC), pH 8.5, stored at room temperature.

3. 10mM Dithiothreitol (DTT) Solution: Prepared in AMBIC buffer.

4. 5mM Iodoacetamide Solution: Freshly prepared in the dark.

5. Trypsin Solution: TPCK-treated trypsin proteomic grade, 1.0 ng/ul in 50 mM AMBIC, pH 8.5, freshly prepared.

Phosphate Group Modification by Using DTT

1. Barium Hydroxide Ba(OH) Solution: 5M in water.

2. Solid Carbonic Dioxide

3. Hepes Buffer Solution: 10mM in water, pH 7.5.

4. DTT Solutions: Light and heavy forms, 30% w/v in Hepes buffer.

Isolation and Enrichment of Tagged Peptides

1. Thiol Sepharose Resin: Pierce Biotechnology, aliquoted.

2. Binding Buffer: 0.1 M Tris-HCl, pH 7.5.

3. Elution Buffer: 20 mM DTT in 10 mM Tris-HCl, pH 7.5.

Mass Spectrometry

1. MALDI Applied Biosystem Voyager DE-PRO Instrument: Operating in reflector mode.

2. MALDI Matrix Solution: o-cyano-hydroxycinnamic acid (10mg/ml) in 70% acetonitrile (ACN), 0.1% trifluoroacetic acid.

3. Peptide Standard Mixture: Applied Biosystems.

4. Single Quadrupole ZQ Electrospray: Coupled to an HPLC.

5. Reverse-phase HPLC Column: Phenomenex 250 x 2.1 mm, 5um.

6. Solvent A: 0.05% TFA, 5% formic acid in water.

7. Solvent B: 95% ACN, 0.05% TFA, 5% formic acid solution.

Phosphate Group Modification by DNS-CL

1. Barium Hydroxide Ba(OH) Solution: 55 M in water.

2. Solid Carbonic Dioxide

3. DANSS Reagent: Prepared by reaction of Dansyl chloride with cystamine.

4. Agilent Zorbax C8 Column: 150 × 4.6mm, 5um.

5. Solvent A and Solvent B: For HPLC.

6. Tris/HCl Buffer: 10mM, pH 8.5.

7. Tributylphosphine Solution: 20mM by dilution in water.

Mass Spectrometry for DNS-CL

1. MALDI-TOF Voyager DE-PRO Mass Spectrometer: Applied Biosystem.

2. ZipTip Pipette: Millipore.

3. Wetting, Equilibration, Washing, and Elution Solutions

4. Matrix Solution: o-cyano-hydroxycinnamic acid in ACN and water.

5. Peptide Standard Mixture: Applied Biosystem.

6. 4000Q-Trap Mass Spectrometer: Applied Biosystems.

7. Agilent Reverse-phase Pre-column Cartridge and Column: Zorbax 300 SB-C18.

8. Uncoated Silica Tip: NewObjectives.

Procedure of Phosphoproteomes Analysis by Selective Labelling and Advanced Mass Spectrometric Techniques

DTT Labelling

Protein Digestion

To ensure efficient digestion and subsequent analysis, precise steps were followed:

• Alkylation: α-casein aliquots were meticulously treated with 5 mM iodoacetamide, promoting alkylation of cysteine residues. This step, crucial for preventing disulfide bond formation and maintaining protein integrity, was carried out under light-protected conditions to prevent unwanted reactions.
• Enzymatic Digestion: A carefully calibrated enzymatic digestion process ensued, employing trypsin solution in a buffered environment (50 mM ammonium bicarbonate, pH 8.5) at a controlled temperature of 37°C for an extended period of 18 hours. This prolonged incubation period facilitated thorough proteolytic cleavage, ensuring maximum peptide yield.
• MALDI-MS Analysis: Following digestion, peptide mixtures were subjected to MALDI-MS analysis in reflector mode. Meticulous attention was paid to instrument parameters, including accelerating voltage, grid settings, and mass range, to optimize spectral acquisition and resolution. External peptide standards were employed for precise mass calibration, enabling accurate identification of phosphopeptides based on characteristic mass shifts.

Reaction β-Elimination

The removal of phosphate moieties from phosphoserine (pSer) and phosphothreonine (pThr) residues was executed with precision:

• Phosphate Removal: β-elimination reactions were mediated by barium hydroxide ions, a robust method for selectively cleaving phosphoester bonds under controlled conditions. The peptide mixture was incubated in a highly concentrated Ba(OH)2 solution at 37°C, followed by neutralization to halt the reaction.
• MALDI-MS Analysis: Analysis of the resulting peptide mixture via MALDI-MS revealed a distinct series of signals corresponding to β-eliminated peptides. This step provided critical insights into the efficacy of phosphate removal and subsequent peptide modification.

DTT as a Bifunctional Reagent

The utilization of dithiothreitol (DTT) as a bifunctional reagent for site-specific modification was meticulously executed:

• Michael-Type Addition: The β-eliminated peptide mixture underwent Michael-type addition with DTT, resulting in the creation of a free thiol group in place of the phosphate moiety. Reaction conditions, including temperature, duration, and reagent concentration, were meticulously optimized to ensure maximal conversion efficiency.
• Monitoring Reaction: The extent of the addition reaction was monitored via MALDI-MS analysis, allowing for precise quantification of DTT-modified peptides. Yield assessment provided valuable insights into reaction kinetics and efficiency, guiding protocol optimization for subsequent experiments.

Affinity Capture of Thiolated Peptides

Selective enrichment of thiolated peptides was achieved through a systematic affinity capture approach:

• Enrichment: Thiolated peptides were specifically captured using thiol sepharose resin, leveraging the affinity of thiol groups for the resin matrix. Stringent washing steps were employed to remove nonspecifically bound peptides, ensuring high purity of the enriched peptide fraction.
• Analysis: The enriched peptide mixture underwent comprehensive analysis via MALDI-MS, enabling precise identification of modified phosphopeptides. This step facilitated the characterization of site-specific phosphorylation events, enhancing our understanding of phosphoprotein dynamics.

Differential Isotope Coded Analysis

Quantitative analysis of DTT-modified peptides was performed using a differential isotope coding strategy:

• Labeling: Stoichiometrically balanced mixtures of α-casein peptides were labeled with light and heavy forms of DTT, allowing for differential quantification of peptide abundance. Varied labeling ratios facilitated accurate determination of relative peptide concentrations.
• LC-MS Analysis: Labeled peptide mixtures were subjected to LC-MS analysis, enabling high-resolution separation and quantification of DTT-modified peptides. Advanced mass spectrometric techniques, coupled with precise chromatographic separation, facilitated robust quantitative analysis of phosphoproteome dynamics.

Dansyl Labelling

α-Casein Digestion

Prior to phosphopeptide analysis, α-casein underwent enzymatic digestion to generate peptide fragments for subsequent labeling and analysis:

• Enzymatic Digestion: α-Casein was digested with trypsin in a buffered solution (50 mM ammonium bicarbonate, pH 8.5) at an optimized enzyme-to-substrate ratio. The digestion process, conducted at 37°C overnight, ensured comprehensive cleavage of peptide bonds, yielding a complex peptide mixture representative of the original protein.
• Sample Preparation: Following digestion, the trypsin digest was dried under vacuum and stored under appropriate conditions to preserve peptide integrity for downstream analysis.

Phosphate Group Modification

Phosphate moieties present on phosphoserine (pSer) and phosphothreonine (pThr) residues were selectively removed prior to dansyl labeling:

• Phosphate Removal: Barium hydroxide ion-mediated β-elimination was employed to cleave phosphate ester bonds, rendering phosphopeptides amenable to subsequent chemical modification. The resulting peptide mixture underwent purification using a ZipTip pipette to remove excess reagents and impurities, ensuring optimal purity for downstream reactions.
• DANS Modification: The β-eliminated peptide mixture was subjected to Michael-type addition with DANSH, introducing dansyl moieties at the site of phosphate removal. This chemical derivatization step endowed peptides with distinctive properties conducive to enhanced ionization and fragmentation during mass spectrometric analysis.

Selective Isolation of Tagged Peptides

To selectively isolate and identify dansyl-labeled phosphopeptides, a multi-step purification and analysis protocol was meticulously executed:

• Peptide Mixture Preparation: A peptide mixture comprising standard proteins and dansyl-cysteamine-modified α-casein peptides was prepared for analysis. This composite mixture served as a representative sample for assessing the feasibility of the labeling strategy in proteomic analysis.
• LC-MS Analysis: The peptide mixture was subjected to LC-MS analysis using a hybrid ion trap mass spectrometer coupled to a nano HPLC system. Precise chromatographic separation facilitated the selective elution of phosphopeptides, while advanced mass spectrometric techniques enabled comprehensive characterization of labeled peptides.
• Data Acquisition and Processing: Spectral data generated during LC-MS analysis were acquired and processed using dedicated software tools. Reconstruction of ion chromatograms, precursor ion scans, and MS2/MS3 spectra facilitated the identification and quantification of dansyl-labeled phosphopeptides, providing valuable insights into phosphoprotein dynamics and site-specific phosphorylation events.

Reference

1. de Graauw, Marjo. Phospho-Proteomics. Humana Press, 2009.