Protocol for Analyzing Phosphoproteomes with Selective Labelling and Advanced Mass Spectrometry

Phosphorylation, a prevalent post-translational modification (PTM) in proteins, plays a crucial role in regulating various cellular processes, including signal transduction, cell cycle progression, and metabolic maintenance. Traditional methods for phosphoproteome analysis, such as radioactive labelling and antibody-based assays, have limitations in terms of sensitivity, specificity, and throughput. Therefore, there is a growing need for more advanced techniques to comprehensively study protein phosphorylation dynamics.

Why Choose Selective Labelling and Advanced Mass Spectrometric Techniques?

Enhanced Sensitivity: Advanced mass spectrometry (MS) platforms, such as Orbitrap and quadrupole-time-of-flight (Q-TOF) instruments, provide high sensitivity, enabling the detection of low-abundance phosphopeptides even in complex samples. This is particularly crucial given the substoichiometric nature of many phosphorylation events.

Increased Specificity: Selective labelling strategies, such as chemical derivatization or affinity tagging, target specific phosphorylation sites or residues, reducing background noise and increasing the specificity of phosphopeptide detection. This ensures accurate identification and quantification of phosphorylated proteins.

Comprehensive Analysis: Advanced MS techniques, such as tandem MS (MS/MS) and electron transfer dissociation (ETD), enable comprehensive characterization of phosphopeptides. MS/MS allows for the fragmentation and sequencing of peptides, facilitating precise localization of phosphorylation sites and determination of phosphorylation stoichiometry.

Quantitative Analysis: Selective labelling combined with stable isotope labelling techniques, such as SILAC (Stable Isotope Labeling by Amino acids in Cell culture) or iTRAQ (Isobaric Tags for Relative and Absolute Quantitation), allows for quantitative analysis of phosphoproteomes. This enables comparative studies of phosphorylation dynamics under different conditions or treatments, providing insights into cellular signaling pathways.

High Throughput: Recent advancements in MS instrumentation, such as high-resolution mass spectrometers and improved data acquisition strategies, have significantly increased the throughput of phosphoproteome analysis. Combined with automated sample preparation and data analysis pipelines, selective labelling coupled with MS enables high-throughput analysis of phosphoproteomes, making it feasible to study large-scale phosphorylation events across multiple samples.

Material

1. Sample Preparation:

• α-Casein Water Solution (1 μg/μl): Store aliquots at -20°C.
• Buffer Solution: 0.1 M Ammonium Bicarbonate (AMBIC), pH 8.5.

2. Reduction and Alkylation:

• 10 mM Dithiothreitol (DTT) Solution in AMBIC Buffer.
• 5 mM Iodoacetamide Solution in AMBIC Buffer: Freshly prepared in the dark.

3. Enzymatic Digestion:

• Trypsin Solution (TPCK-treated Trypsin Proteomic Grade): 1.0 ng/μl in 50 mM AMBIC, pH 8.5. Freshly prepared.

4. Phosphopeptide Enrichment:

• 1 ml Thiol Sepharose Resin: Pierce Biotechnology; Rockford, IL (aliquoted).
• Binding Buffer: 0.1 M Tris-HCl, pH 7.5.
• Elution Buffer: 20 mM DTT in 10 mM Tris-HCl, pH 7.5.

5. Mass Spectrometric Analysis:

• MALDI Applied Biosystem Voyager DE-PRO Instrument operating in reflector mode.
• MALDI Matrix Solution: α-cyano-hydroxycinnamic acid (10 mg/ml) in 70% acetonitrile (ACN), 0.1% trifluoroacetic acid.
• Peptide Standard Mixture (Applied Biosystems).

6. Additional Materials for Specific Techniques:

For Barium Hydroxide Ba(OH)2 Method:

• Barium Hydroxide Ba(OH)2 Solution: 55 M in Water.
• Solid Carbonic Dioxide.
• Hepes Buffer Solution: 10 mM in Water, pH 7.5.
• DTT Solutions: Light and heavy (CEA; Saclay, France) form, 30% w/v in Hepes buffer.

For DANSS Method:

• DANSS Reagent: Prepared by reaction of Dansyl chloride (0.1 mg/ml dissolved in ACN) with cystamine (molar ratio 3:1).
• Agilent Zorbax C8 Column (150 × 4.6 mm2 i.d.).
• Solvent A: 0.1% formic acid, 2% ACN in water.
• Solvent B: 0.1% formic acid, 2% water in ACN.
• 10 mM Tris/HCl Buffer, pH 8.5.
• 20 mM Tributylphosphine Solution: Diluted in water from the stock solution (200 mM), freshly prepared.
• DANSH Solution: 0.1 mg/ml, dissolved in ACN/water 3:1. Freshly prepared.
• 1 mM Tributylphosphine Solution in Tris 2 M Solution, pH 10.8.

For MALDI-TOF MS Method:

• MALDI-TOF Voyager DE-PRO Mass Spectrometer (Applied Biosystem, Framingham, MA).
• ZipTip Pipette from Millipore (Billerica, MA) using the recommended purification procedure.
• Wetting Solution: 50% ACN in Water.
• Equilibration and Washing Solutions: 0.1% TFA.
• Elution Solution: 50% ACN, 0.1% TFA in Water.
• Peptide Standard Mixture from Applied Biosystem.
• Matrix Solution: α-cyano-hydroxycinnamic acid (10 mg/ml in 70% ACN and 0.1% TFA in water).

For ESI-MS Method:

• 4000Q-Trap (Applied Biosystems) Mass Spectrometer equipped with a linear ion trap coupled to 1100 nano HPLC system (Agilent Technologies).
• Agilent Reverse-phase Pre-column Cartridge (Zorbax 300 SB-C18, 5 × 0.3 mm2, 5 μm).
• Agilent Reverse-phase Column (Zorbax 300 SB-C18, 150 mm × 75 μm, 3.5 μm).
• Solvent A: 0.1% formic acid, 2% ACN in water.
• Solvent B: 0.1% formic acid, 2% water in ACN.
• Uncoated Silica Tip from NewObjectives (Ringoes, NJ) (O.D. 150 μm, i.d. 20 μm, tip diameter 10 μm).

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

1. Reduction and Alkylation:

• Alkylate one aliquot of α-casein with 5 mM iodoacetamide for 30 min at room temperature in the dark.
• Digest the alkylated α-casein with trypsin solution in 50 mM ammonium bicarbonate pH 8.5 at 37°C for 18 h.

2. Phosphorylation Site Identification:

• Analyze the peptide mixtures by MALDI-MS in reflector mode.
• Remove phosphate moieties via barium hydroxide ion-mediated β-elimination from phosphoserine (pSer) and phosphothreonine (pThr) residues.
• Analyze the resulting peptide mixture by MALDI-MS.
• Monitor the extent of the reaction via MALDI-MS.

3. Thiol Group Addition and Enrichment:

• Add dithiothreitol (DTT) in the light and heavy form to the β-eliminated peptide mixtures.
• Carry out the addition reaction for 3 h at 50°C under nitrogen.
• Enrich thiolated peptides using activated thiol sepharose resin.
• Wash and extract modified peptides using elution buffer.
• Dissolve the enriched peptide mixture for MALDI-MS analysis.

4. Isotope Tagging and Quantification:

• Label samples containing stoichiometric concentrations of α-casein with DTT and DTT-D6 in various ratios.
• Analyze the peptide mixtures by MALDI-MS in reflector mode.
• Quantify the peptides by measuring the relative signal intensities for pairs of peptide ions differentially labeled with light or heavy DTT.
• Analyze aliquots of the peptide mixtures by LC-MS.

5. Dansyl Labelling and Analysis:

• Digest α-casein with trypsin and remove phosphate moieties via barium hydroxide ion-mediated β-elimination.
• Analyze the peptide mixture by MALDI-MS.
• Purify the peptide mixtures and modify with DANSH via Michael-type addition.
• Analyze the modified peptides by ESI-MS.
• Dissolve dried DANSS in Tris buffer and reduce in the presence of tributylphosphine.
• Purify the product by RP-HPLC and verify by ESI-MS.
• Label the β-eliminated peptide mixture with DANSH and analyze the reaction yield via MALDI-MS.

6. Application to Proteomic Analysis:

• Prepare a peptide mixture from standard proteins and spike with α-casein trypsin mixture modified with dansyl-cysteamine.
• Submit the peptide mixture to LC-MS analysis.
• Separate peptides on a reverse-phase column and acquire spectra.
• Analyze and process data using appropriate software.

Reference

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