A GTP affinity probe for proteomics highlights flexibility in purine nucleotide selectivity

Area of Science:

• Biochemistry
• Proteomics
• Molecular Biology

Background:

• GTP-binding proteins (GTP-BPs) are crucial in cellular processes.
• Proteomic profiling is effective for studying diverse protein families.
• Existing methods may not identify unknown GTP-BP targets.

Purpose of the Study:

• To synthesize and characterize a novel photoreactive GTP affinity probe.
• To enable identification of GTP-binding proteins using click chemistry and mass spectrometry.
• To investigate nucleotide selectivity of identified proteins.

Main Methods:

• Synthesis of a photoreactive GTP affinity probe (GTP-BP-yne) with an alkyne handle.
• Photocross-linking of the probe to cellular proteins.
• Click chemistry conjugation to reporter tags.
• Mass spectrometry-based proteomic profiling for target identification.

Main Results:

• The GTP-BP-yne probe successfully identified various GTP-binding proteins, including small GTPases and GTP1/OBG family members.
• ATP-binding proteins were also identified, indicating variable purine nucleotide selectivity.
• The probe facilitated the elucidation of relative nucleotide selectivities for identified targets.

Conclusions:

• The GTP-BP-yne probe is a valuable tool for studying GTP-binding proteins.
• It is particularly useful for identifying unknown targets in proteomic studies.
• The probe aids in understanding protein nucleotide selectivity.