Experimental Techniques in Biochemistry

Mass Spectrometry:
- Identifies and quantifies posttranslational modifications (PTMs) by analyzing mass-to-charge ratios.
- Can detect multiple modifications and their sites on proteins.
Western Blotting:
- Used for detection and quantification of specific proteins with PTMs using antibodies.
- Modification-specific antibodies enhance sensitivity for detecting PTMs.
Chromatography:
- Separates modified proteins based on size, charge, or hydrophobicity.
- High-Performance Liquid Chromatography (HPLC) is commonly used for purification prior to mass spectrometry.
Nuclear Magnetic Resonance (NMR):
- Provides structural information about proteins and their modifications in solution.
CRISPR/Cas9 Systems:
- Gene editing tools that can be used to introduce or remove specific PTMs in proteins.

Phosphorylation:
- Addition of phosphate groups; regulates activity, localization, and interaction with other proteins.
Glycosylation:
- Attachment of carbohydrate moieties; affects protein stability, folding, and recognition by other molecules.
Acetylation:
- Addition of acetyl groups, primarily on lysine residues; influences gene expression and protein function.
Ubiquitination:
- Attachment of ubiquitin molecules; tags proteins for degradation via the proteasome.
Methylation:
- Addition of methyl groups, commonly on lysine and arginine residues; modifies protein interaction and stability.
Sumoylation:
- Addition of small ubiquitin-like modifier (SUMO); alters protein stability and localization.
Fatty Acylation:
- Addition of lipid groups; affects membrane association and signaling.

Regulation of Activity:
- PTMs can activate or deactivate enzymes and receptors, modulating metabolic pathways.
Protein Stability:
- Modifications can protect proteins from degradation or mark them for destruction.
Intracellular Localization:
- PTMs can influence the subcellular distribution of proteins.
Protein-Protein Interactions:
- Modifications can create or disrupt binding sites, affecting signaling cascades.
Response to Environmental Signals:
- PTMs enable rapid cellular responses to stress, signaling, and developmental cues.

Kinases:
- Enzymes that catalyze the addition of phosphate groups (phosphorylation).
Phosphatases:
- Remove phosphate groups, reversing the action of kinases.
Glycosyltransferases:
- Enzymes that attach carbohydrates to proteins (glycosylation).
Acetyltransferases and Deacetylases:
- Add or remove acetyl groups, respectively (acetylation and deacetylation).
Ubiquitin Ligases:
- Catalyze the attachment of ubiquitin to target proteins (ubiquitination).
Methyltransferases and Demethylases:
- Add or remove methyl groups from proteins (methylation).
SUMO Ligases and Proteases:
- Responsible for the addition (sumoylation) and removal (de-sumoylation) of SUMO moieties.

Mass Spectrometry: Identifies posttranslational modifications (PTMs) through mass-to-charge ratio analysis; effective in detecting various modifications simultaneously.
Western Blotting: A method to detect specific proteins with PTMs utilizing antibodies; modification-specific antibodies increase the assay's sensitivity.
Chromatography: Separates proteins based on properties like size, charge, or hydrophobicity; High-Performance Liquid Chromatography (HPLC) is often utilized for purification before mass spectrometry.
Nuclear Magnetic Resonance (NMR): Offers structural insights into proteins and their modifications while in solution.
CRISPR/Cas9 Systems: Advanced gene editing technologies that permit the introduction or removal of specific PTMs within proteins.

Phosphorylation: The process of adding phosphate groups which modulates protein activity, localization, and interactions.
Glycosylation: The addition of carbohydrate moieties that influences protein stability, folding, and molecular recognition.
Acetylation: Involves adding acetyl groups mainly to lysine residues, impacting gene expression and overall protein functionality.
Ubiquitination: The attachment of ubiquitin molecules serves to tag proteins for proteasomal degradation.
Methylation: Involves the addition of methyl groups, mainly on lysine and arginine residues, modifying protein stability and interaction.
Sumoylation: The addition of small ubiquitin-like modifiers (SUMO) affects a protein's stability and localization.
Fatty Acylation: The process of adding lipid groups alters membrane affinity and cellular signaling pathways.

Regulation of Activity: PTMs enable the activation or inhibition of enzymes and receptors, thus fine-tuning metabolic processes.
Protein Stability: Modifications can either shield proteins from degradation or signal them for breakdown.
Intracellular Localization: PTMs can determine the specific cellular locations where proteins function.
Protein-Protein Interactions: Modifications play a critical role in creating or disrupting interactions between proteins, facilitating various signaling pathways.
Response to Environmental Signals: PTMs allow cells to swiftly react to external stressors or developmental signals, ensuring adaptive growth.

Kinases: Enzymes that facilitate the addition of phosphate groups, driving phosphorylation events.
Phosphatases: Responsible for removing phosphate groups, counteracting the function of kinases.
Glycosyltransferases: Enzymes that catalyze the addition of carbohydrate moieties to proteins, enabling glycosylation.
Acetyltransferases and Deacetylases: Enzymes that add or remove acetyl groups, playing roles in acetylation and deacetylation processes.
Ubiquitin Ligases: Catalysts for the addition of ubiquitin to target proteins, initiating ubiquitination.
Methyltransferases and Demethylases: Enzymes that facilitate the addition or removal of methyl groups, thus regulating methylation status.
SUMO Ligases and Proteases: Enzymes managing the sumoylation process by adding or removing SUMO moieties from target proteins.