The human genome comprises approximately 20,000 to 25,000 protein-coding genes. However, the number of protein species in the human body is far greater than the number of genes, estimated to be between 200,000 and 2 million. The generation of diversity from gene to mRNA to protein is a multi-tiered and multi-mechanistic process. These mechanisms include genetic-level variations (such as mutations, fusions, and duplications), transcriptional regulation (such as alternative splicing, selection of transcription start sites, selection of transcription termination sites, and regulation by non-coding RNAs), and post-translational modifications (such as phosphorylation, ubiquitination, acetylation, glycosylation, lipidation, and oxidative modifications). These mechanisms work in concert to greatly enrich the structural and functional diversity of proteins, thereby supporting the complex physiological functions and adaptability of organisms.

 

1. Definition and Overview of Protein Post-translational Modifications

 

Protein post-translational modifications (PTMs) refer to the covalent addition of various chemical groups to amino acid residues or the processing of proteins through cleavage and folding, following protein synthesis. These modifications alter the structure, stability, activity, localization, and interaction capabilities of proteins with other molecules. PTMs typically occur in cellular compartments such as the cytoplasm, endoplasmic reticulum, and Golgi apparatus, and are catalyzed by a series of highly specific enzymes. A wide array of PTM types has been identified, including phosphorylation, ubiquitination, acetylation, methylation, glycosylation, ubiquitin-like protein modifications, lipidation, and oxidative modifications. Each type of modification has its unique biological significance and mechanism of action, and they work in concert to form a complex and intricate regulatory network that precisely controls protein function and cellular physiology.

 

 

2. Common Types of Protein Post-translational Modifications and Their Functions

 

2.1Phosphorylation

Phosphorylation is one of the most common post-translational modifications of proteins, primarily occurring on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues. This modification is typically catalyzed by protein kinases, which transfer phosphate groups from ATP molecules to specific amino acid residues, thereby altering the structure and function of the protein. Phosphorylation plays a central role in cellular signal transduction, regulating protein activity, stability, subcellular localization, and interactions with other proteins. For example, many key signaling pathways involved in cell growth, differentiation, apoptosis, and metabolic regulation rely on phosphorylation to transmit and amplify signals. When extracellular signaling molecules (such as hormones or growth factors) bind to cell surface receptors, they activate a cascade of protein kinase reactions. Through phosphorylation, the signal is relayed from the receptor to the interior of the cell, ultimately leading to changes in the activity of intracellular target proteins and triggering the corresponding cellular responses. Additionally, phosphorylation is involved in the regulation of gene expression; some transcription factors, upon phosphorylation, can bind to DNA and modulate transcriptional activity. As one of the most extensively studied types of post-translational modifications, antibodies for the detection of phosphorylated proteins are also relatively common.

 

2.2 Ubiquitination

Ubiquitination is the process by which ubiquitin molecules are covalently attached to target proteins, primarily accomplished through the coordinated action of ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). Ubiquitination plays a crucial role in various biological processes, including protein degradation, cell cycle regulation, DNA repair, and signal transduction. The most typical function is the formation of polyubiquitin chains, which mark target proteins for degradation via the proteasome pathway, thereby regulating protein levels and maintaining cellular homeostasis. For example, during different stages of the cell cycle, many cyclins are degraded by the proteasome through ubiquitination, ensuring the proper progression of the cell cycle. In addition, ubiquitination can also regulate the subcellular localization, activity, and interactions of proteins with other molecules, participating in the regulation of multiple intracellular signaling pathways, such as the NF-κB signaling pathway and the Wnt signaling pathway.

 

 

2.3 Acetylation

Acetylation is a post-translational modification that predominantly occurs on lysine residues, catalyzed by acetyltransferases that transfer the acetyl group from acetyl-CoA to the amino group of lysine. This modification plays a significant role in various biological processes, including the regulation of gene expression, chromatin remodeling, cellular metabolism, and protein stability.

 

In the context of gene expression regulation, histone acetylation is a key epigenetic modification. The acetylation of histones neutralizes their positive charges, thereby weakening the interaction between histones and DNA and loosening the chromatin structure. This facilitates the binding of transcription factors and RNA polymerase to DNA, thereby activating gene transcription. In addition to histones, many non-histone proteins are also subject to acetylation. These modifications can regulate protein activity, stability, subcellular localization, and interactions with other proteins, participating in a variety of metabolic processes and signaling pathways within the cell.

 

Lysine Acetylation

 

2.4 Glycosylation

Glycosylation is the process of attaching carbohydrate groups to proteins and is primarily classified into two types: N-glycosylation and O-glycosylation. N-glycosylation occurs on asparagine residues, while O-glycosylation mainly takes place on serine, threonine, or hydroxylysine residues. Glycosylation plays a crucial role in protein folding, stability, subcellular localization, intercellular recognition, and signal transduction.

 

During protein folding, glycosylation can act as a molecular chaperone, assisting in the correct folding of proteins and preventing protein aggregation. Glycosylation can also modulate protein stability and influence intracellular trafficking and subcellular localization. In terms of intercellular recognition and signal transduction, glycoproteins on the cell surface interact with extracellular ligands, receptors, or the extracellular matrix through their glycosylation modifications, participating in processes such as cell–cell adhesion, immune recognition, and cell migration. For example, in the immune system, the glycosylation of antibodies can affect their binding affinity to antigens and their immunological effector functions.

 

N-glycosylation and O-glycosylation

 

3. Regulatory Mechanisms of Protein Post-translational Modifications

 

The regulation of protein post-translational modifications (PTMs) is a complex process involving the interplay of multiple factors. First and foremost, the activities of modifying enzymes and de-modifying enzymes are key determinants in regulating PTMs. The expression levels, activity states, and substrate binding affinities of these enzymes all influence the extent of PTMs. For instance, the balance of activities between protein kinases and phosphatases dictates the phosphorylation levels of proteins, while the equilibrium between acetyltransferases and deacetylases determines the acetylation levels. Secondly, intracellular signaling pathways also modulate PTMs. Extracellular signaling molecules, by activating specific signaling cascades, can regulate the activities of modifying and de-modifying enzymes, thereby altering the PTM status of proteins. For example, the PI3K-Akt signaling pathway activated by growth factors can modulate the activity of protein kinases, subsequently affecting phosphorylation modifications. Additionally, the intracellular metabolic and redox states can also impact PTMs. For example, the cellular energy metabolism status can influence the levels of acetyl-CoA, thereby regulating the degree of acetylation modifications; oxidative stress can induce oxidative modifications of proteins, such as tyrosine nitration and cysteine oxidation.

 

4. Research Methods and Techniques for Protein Post-translational Modifications

 

With the continuous advancement of biotechnology, the methods and techniques for studying protein post-translational modifications (PTMs) have become increasingly diverse. Traditional approaches include immunoprecipitation, Western blotting, and mass spectrometry. Immunoprecipitation and Western blotting techniques can be employed to detect the PTM status of specific proteins. These methods rely on specific antibodies to recognize and bind to the modified proteins, thereby enabling qualitative and quantitative analyses of protein modifications.

 

In recent years, with the rapid development of proteomics technologies, mass spectrometry-based proteomics has emerged as a powerful tool for studying PTMs. Through high-throughput proteomic analyses, it is now possible to simultaneously identify and quantify the PTM states of a large number of proteins within cells or tissues. This approach allows for the elucidation of global changes in protein modifications and the underlying regulatory networks.

 

WB Example：

 

WB resuLt of Phospho-NF-kB p105/p50 (Ser337) Recombinant Rabbit mAb

Primary antibody: Phospho-NF-kB p105/p50 (Ser337) Recombinant Rabbit mAb at 1/5000 dilution
Lane 1: untreated NIH/3T3 whole cell lysate 20 µg
Lane 2: NIH/3T3 treated with 20 ng/mL TNF-alpha and 100 nM CalycuLin A for 10 minutes whole cell lysate 20 µg
Secondary antibody: Goat Anti-Rabbit IgG, (H+L), HRP conjugated at 1/10000 dilution Predicted MW: 105 kDa
Observed MW: 55 kDa

 

CoIP Example：

Either enrich the total protein using an antibody specific to the target protein and then detect the post-translational modifications of the enriched protein with a pan-modification antibody, or enrich all proteins bearing a specific modification using a pan-modification antibody and subsequently detect the post-translational modifications of the target protein with an antibody specific to that protein.

 

 

Endogenous and exogenous immunoprecipitation experiments were conducted in MDA-MB-231 and HEK293T cells using plasmid transfection. The results showed that specific lactylation bands were detected at the molecular weight of RCC2 in both endogenous and exogenous immunoprecipitates (Figures D and E).

 

References：

[1]Luo C, Xu H, Yu Z, Liu D, Zhong D, Zhou S, Zhang B, Zhan J, Sun F. Meiotic chromatin-associated HSF5 is indispensable for pachynema progression and male fertility. Nucleic Acids Res. 2024 Sep 23;52(17):10255-10275. doi: 10.1093/nar/gkae701. PMID: 39162221; PMCID: PMC11417359.

 

CatalogNumber	Product Name	Specification
abs955	Immunoprecipitation (IP) / Co-Immunoprecipitation (CoIP) Kit	50T
abs9649	rProtein A/G Magnetic IP/Co-IP Kit	50T
abs160027	Anti-GST Agarose Beads	125uL(5reactions)
abs160026	Anti-Myc Tag Agarose Beads	125uL(5reactions)
abs160022	Anti-RFP/mcherry Agarose Beads	125uL(5reactions)
abs111870	Rabbit anti-5-hydroxymethylcytosine Polyclonal Antibody	50uL
abs118910	Rabbit anti-Acetyl-Histone H3-K27 Polyclonal Antibody	50uL
abs149356	Rabbit anti-Histone H2A(Butyryl-K5) Polyclonal Antibody	50uL
abs149363	Rabbit anti-Histone H3(MonoMethyl-K9) Polyclonal Antibody	50uL
abs20213	Mouse anti-Rabbit IgG-HRP Antibody(Conformation Specific)	50uL
abs20212	Rat anti-Mouse lgG-HRP Antibody(Conformation Specific)	50uL

 

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