H1: Post translational modification - phosphoproteomics
Enrichement of phosphopeptides
Immuno A3inity Chromatography (IAC) Immuno A3inity Chromatography (IAC)
• Technique to selecively capture and isolate specific proteins or peptides based on their antigenic properties
• Using antibodies (Ab)
Antibodies to capture phosphorylated peptides
• Ab against pY (phosphotyrosine), pS (phosphoserine), and pT (phosphothreonine) are commonly used
• pS and pT antibodies: less specific due to potential cross-reactivity with other acidic AA (like Asp and Glu).
• Ab against kinase consensus sequences can also be used for broader specificity.
Applications:
• Often used in combination with other methods like IMAC (immobilized metal aKinity chromatography) or
• TiO2 (titanium dioxide chromatography) to enhance specificity and capture phosphorylated peptides more
eKiciently.
Immobilized Metal A3inity chromatography (IMAC) This technique takes advantage of the aKinity between negatively charged phosphate groups on peptides and metal ions
immobilized on a stationary phase.
Preparation of the Stationary Phase:
• Metal Chelation: Metal ions (e.g., Fe3+, Ga3+) are chelated (chemically bound) to nitrilotriacetic acid (NTA) ligands,
which are pre-attached to beads.
• This creates a stable metal-NTA complex that forms the stationary phase for chromatography.
Binding of Phosphopeptides:
• Phosphopeptides have (-) charged phosphate groups that interact strongly with the (+) charged metal ions.
• The binding is driven by the electrostatic interaction between the phosphate groups and the metal centers.
• Non-specific Binding: Other acidic peptides (rich in aspartic or glutamic acid residues) or non-phosphorylated
peptides with anionic features may also bind to some extent, reducing specificity.
Preference for Multiphosphorylated Peptides:
• Peptides with multiple phosphate groups exhibit stronger binding due to their higher negative charge and greater
aKinity for the stationary phase.
Elution of Bound Peptides:
• Elution is achieved under basic conditions (high pH), which disrupts the interaction between the phosphate
groups and the metal ions.
1
, Sequential IMAC (SIMAC) • SIMAC = variation of IMAC to enhance specificity
• Specifically designed for the stepwise enrichment of phosphorylated peptides.
• By sequentially eluting peptides under diKerent conditions à separation into distinct fractions
• Non-phosphorylatedpeptides
• Monophosphorylatedpeptides
• Multiphosphorylatedpeptides
Workflow and fractions
Sample Loading:
• The peptide mixture is applied to IMAC column
• Phosphorylated peptides bind to the stationary phase through their (-) charged phosphate groups, while some
non-phosphorylated or acidic peptides may bind weakly or remain in the flow-through.
Fraction 1: Flow-Through:
• Contains primarily non-phosphorylated peptides and some phosphorylated peptides that do not bind
strongly to the IMAC column.
• These peptides can be discarded or analyzed separately
Fraction 2: Acidic Elution (Less Specific):
• Elution Method: A mildly acidic solution (e.g., pH ~3) is used to disrupt weaker interactions between the column
and peptides.
• Contains a mix of non-phosphorylated peptides and (mono)phosphorylated peptides, as mono’s typically bind
less strongly to the metal ions compared to multiphosphorylated peptides.
• Further Analysis: This fraction can be subjected to additional purification using a titanium dioxide (TiO2) column,
which has a high aKinity for phosphorylated peptides, to further enrich the monophosphorylated species.
Fraction 3: Basic Elution (Highly Specific):
• Elution Method: Uses a basic buKer (e.g., pH ~9–10) to elute peptides most strongly bound to IMAC column.
• Primarily contains multiphosphorylated peptides, which have stronger binding due to their higher negative charge
and greater aKinity for the metal ions.
2
Enrichement of phosphopeptides
Immuno A3inity Chromatography (IAC) Immuno A3inity Chromatography (IAC)
• Technique to selecively capture and isolate specific proteins or peptides based on their antigenic properties
• Using antibodies (Ab)
Antibodies to capture phosphorylated peptides
• Ab against pY (phosphotyrosine), pS (phosphoserine), and pT (phosphothreonine) are commonly used
• pS and pT antibodies: less specific due to potential cross-reactivity with other acidic AA (like Asp and Glu).
• Ab against kinase consensus sequences can also be used for broader specificity.
Applications:
• Often used in combination with other methods like IMAC (immobilized metal aKinity chromatography) or
• TiO2 (titanium dioxide chromatography) to enhance specificity and capture phosphorylated peptides more
eKiciently.
Immobilized Metal A3inity chromatography (IMAC) This technique takes advantage of the aKinity between negatively charged phosphate groups on peptides and metal ions
immobilized on a stationary phase.
Preparation of the Stationary Phase:
• Metal Chelation: Metal ions (e.g., Fe3+, Ga3+) are chelated (chemically bound) to nitrilotriacetic acid (NTA) ligands,
which are pre-attached to beads.
• This creates a stable metal-NTA complex that forms the stationary phase for chromatography.
Binding of Phosphopeptides:
• Phosphopeptides have (-) charged phosphate groups that interact strongly with the (+) charged metal ions.
• The binding is driven by the electrostatic interaction between the phosphate groups and the metal centers.
• Non-specific Binding: Other acidic peptides (rich in aspartic or glutamic acid residues) or non-phosphorylated
peptides with anionic features may also bind to some extent, reducing specificity.
Preference for Multiphosphorylated Peptides:
• Peptides with multiple phosphate groups exhibit stronger binding due to their higher negative charge and greater
aKinity for the stationary phase.
Elution of Bound Peptides:
• Elution is achieved under basic conditions (high pH), which disrupts the interaction between the phosphate
groups and the metal ions.
1
, Sequential IMAC (SIMAC) • SIMAC = variation of IMAC to enhance specificity
• Specifically designed for the stepwise enrichment of phosphorylated peptides.
• By sequentially eluting peptides under diKerent conditions à separation into distinct fractions
• Non-phosphorylatedpeptides
• Monophosphorylatedpeptides
• Multiphosphorylatedpeptides
Workflow and fractions
Sample Loading:
• The peptide mixture is applied to IMAC column
• Phosphorylated peptides bind to the stationary phase through their (-) charged phosphate groups, while some
non-phosphorylated or acidic peptides may bind weakly or remain in the flow-through.
Fraction 1: Flow-Through:
• Contains primarily non-phosphorylated peptides and some phosphorylated peptides that do not bind
strongly to the IMAC column.
• These peptides can be discarded or analyzed separately
Fraction 2: Acidic Elution (Less Specific):
• Elution Method: A mildly acidic solution (e.g., pH ~3) is used to disrupt weaker interactions between the column
and peptides.
• Contains a mix of non-phosphorylated peptides and (mono)phosphorylated peptides, as mono’s typically bind
less strongly to the metal ions compared to multiphosphorylated peptides.
• Further Analysis: This fraction can be subjected to additional purification using a titanium dioxide (TiO2) column,
which has a high aKinity for phosphorylated peptides, to further enrich the monophosphorylated species.
Fraction 3: Basic Elution (Highly Specific):
• Elution Method: Uses a basic buKer (e.g., pH ~9–10) to elute peptides most strongly bound to IMAC column.
• Primarily contains multiphosphorylated peptides, which have stronger binding due to their higher negative charge
and greater aKinity for the metal ions.
2
