Protein post-translational modifications (PTMs) broadly refer to covalent processing events that occur during or after translation, including proteolytic cleavage and the covalent addition or removal of chemical groups, small molecules, or protein modifiers, thereby regulating protein activity, stability, subcellular localization, and molecular interactions. Phosphorylation, ubiquitination, SUMOylation, glycosylation, methylation, acetylation, lactylation, and other modifications jointly participate in signal transduction, metabolic regulation, gene expression, DNA damage repair, and immune responses .

Keywords: protein post-translational modifications; ubiquitination; SUMOylation; phosphorylation; glycosylation; methylation; acetylation; lactylation; modification proteomics; mass spectrometry analysis

1 Types and Functions of Protein Post-Translational Modifications

1.1 Regulatory Roles of Protein Post-Translational Modifications

Post-translational modifications can alter the charge, steric properties, and chemical characteristics of amino acid residues, thereby affecting protein conformation, enzymatic activity, stability, subcellular localization, and protein interactions. The same protein may undergo multiple modifications at different residues, and different modifications may exhibit cooperative, competitive, or sequential regulation, thereby expanding the structural and functional diversity of the proteome.

1.2 Common Types of Post-Translational Modifications

Protein post-translational modifications include phosphorylation, ubiquitination, SUMOylation, glycosylation, methylation, acetylation, lactylation, S-nitrosylation, lipidation, palmitoylation, myristoylation, carbamylation, and proteolytic processing. Different modifications have distinct substrate residues, catalytic systems, and biological functions.

Figure 1 Common types of protein post-translational modifications and their target residues

Table 1 Major Characteristics of Common Protein Post-Translational Modifications

2 Research Workflow for Modification Proteomics

2.1 Sample Preparation and Protein Digestion

Total proteins are extracted after lysis of tissue, cell, or body-fluid samples and are then reduced, alkylated, and digested with proteases to generate peptide mixtures. During sample preparation, protease inhibitors, phosphatase inhibitors, deubiquitinating enzyme inhibitors, or deacetylase inhibitors should be added according to the target modification to reduce modification loss caused by sample processing.

2.2 Enrichment of Modified Peptides

Most modified peptides are present at low abundance and require selective enrichment before mass spectrometric detection. Phosphopeptides can be enriched by immobilized metal ion affinity chromatography or metal oxide affinity methods; glycopeptides can be enriched using lectins, hydrophilic interaction chromatography, or chemical derivatization; ubiquitination sites can be enriched using antibodies that recognize the diglycine remnant on lysine residues after tryptic digestion; and acetylated, methylated, and lactylated peptides can be enriched using modification-specific antibodies.

2.3 Mass Spectrometric Detection and Site Localization

Liquid chromatography-tandem mass spectrometry can identify modification types according to precursor-ion mass shifts and determine peptide sequences and modification sites using tandem mass spectrometric fragment ions. When the same peptide contains multiple candidate modification residues, site-localization probability, diagnostic fragment ions, and results from replicate samples should be combined to evaluate site confidence.

2.4 Bioinformatics Analysis

After database searching of mass spectrometric data, information on modified peptides, modification sites, and corresponding proteins can be obtained, followed by differential-modification analysis, sequence-motif analysis, protein functional annotation, subcellular-localization analysis, signaling-pathway enrichment, and protein-interaction network analysis. Modification-omics data should also be integrated with total proteomic data to distinguish changes in modification stoichiometry from changes in total protein abundance.

2.5 Phenotypic and Mechanistic Validation

Candidate modification sites can be validated using amino acid mutagenesis, protein knockdown or knockout, treatment with modification-enzyme inhibitors, and recombinant protein experiments. Mechanistic studies can further analyze the effects of modification sites on enzymatic activity, protein stability, subcellular localization, complex formation, and cellular phenotypes.

Table 2 Major Research Stages in Modification Proteomics

3 Databases and Visualization Tools for Post-Translational Modification Research

3.1 Protein and Enzyme Databases

UniProt can be used to query protein sequences, functions, subcellular localization, and known modification sites; PIR provides protein-sequence classification and functional annotation information; and BRENDA is mainly used to query enzymatic reactions, substrates, kinetic parameters, and inhibitors.

3.2 Post-Translational Modification Databases

PhosphoSitePlus integrates information on phosphorylation, ubiquitination, acetylation, methylation, and other modifications of mammalian proteins; PTMD collects evidence linking post-translational modifications with human diseases; and PROSITE can identify functional domains, conserved motifs, and potential functional regions based on protein sequences.

3.3 Protein Structure and Site Visualization

PyMOL and Chimera can be used to display protein three-dimensional structures, the locations of modified residues, and their spatial relationships with ligands or interacting proteins. Modification sites can be mapped onto protein domains, catalytic centers, or molecular interaction interfaces to analyze their potential effects on protein function.

Table 3 Common Databases and Software Used in Post-Translational Modification Research

4 Protein Ubiquitination

4.1 Ubiquitination Reaction Process

Ubiquitination is sequentially catalyzed by ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. E1 uses ATP to activate ubiquitin and forms a thioester bond with the C terminus of ubiquitin; activated ubiquitin is then transferred to a cysteine residue of E2; and E3 recognizes the substrate protein and promotes attachment of ubiquitin to lysine or another acceptor site on the substrate.

4.2 Ubiquitin-Chain Types and Their Functions

Substrate proteins may undergo monoubiquitination, multisite monoubiquitination, or polyubiquitination. K48-linked polyubiquitin chains mainly participate in proteasomal degradation, whereas K63-linked ubiquitin chains mainly participate in signal transduction, membrane trafficking, DNA damage repair, and autophagy regulation. Ubiquitin can also form different chain types through K6, K11, K27, K29, K33, M1, and other linkage sites.

4.3 Regulation by Deubiquitination

Deubiquitinating enzymes can remove ubiquitin from substrate proteins or edit ubiquitin-chain structures, making ubiquitination a dynamic and reversible regulatory process. Abnormalities in ubiquitin ligases, deubiquitinating enzymes, or proteasomal activity can disrupt protein homeostasis and affect the cell cycle, apoptosis, immunity, and tumor-related signaling.

4.4 Methods for Ubiquitination Research

Ubiquitination can be studied using activity-based probes to detect the activities of ubiquitination enzymes or deubiquitinating enzymes, while ubiquitin tags, immunoprecipitation-immunoblotting, and mass spectrometric analysis can be used to evaluate ubiquitination of target proteins. Ubiquitinomics generally combines enrichment of diglycine-remnant peptides with high-resolution mass spectrometry to systematically analyze ubiquitination sites and their changes.

5 Protein SUMOylation

5.1 SUMO Maturation and Transfer

SUMO precursors are first processed by SUMO-specific proteases to expose the C-terminal diglycine motif and form mature SUMO. Mature SUMO is activated by E1 in an ATP-dependent manner and transferred to E2 Ubc9, which can directly catalyze formation of an isopeptide bond between SUMO and a substrate lysine; SUMO E3 ligases enhance substrate selectivity and conjugation efficiency in many reactions but are not essential for all SUMOylation events .

5.2 Regulation by DeSUMOylation

SENP-family proteins participate in both SUMO-precursor processing and removal of SUMO from substrate proteins. The dynamic balance between SUMO conjugation and deSUMOylation determines the modification level, subcellular localization, and functional state of substrate proteins.

5.3 Biological Functions of SUMOylation

SUMOylation mainly participates in protein stability, nucleocytoplasmic transport, transcriptional regulation, chromatin organization, cell-cycle regulation, and DNA damage repair. SUMOylation generally does not directly mediate proteasomal degradation but can affect substrate stability by altering protein interactions or through crosstalk with the ubiquitin system.

6 Protein Phosphorylation

6.1 Phosphorylation and Dephosphorylation

Canonical eukaryotic serine/threonine and tyrosine kinases mainly transfer the γ-phosphate group of ATP to serine, threonine, or tyrosine residues on substrate proteins, whereas residues such as histidine and aspartate can also undergo noncanonical phosphorylation. The phosphate group can alter local charge, protein conformation, and molecular-binding capacity, whereas protein phosphatases terminate or readjust signaling by removing phosphate groups.

6.2 Biological Functions of Phosphorylation

Phosphorylation can rapidly regulate enzymatic activity, receptor signaling, and protein interactions and participates in cell proliferation, differentiation, metabolism, the cell cycle, apoptosis, and immune responses. Consecutive kinase and phosphatase reactions can form signaling cascades, feedback regulation, and pathway crosstalk.

6.3 Methods for Phosphorylation Research

Phosphorylation can be studied using kinase activity assays, phosphatase treatment, radioactive ATP labeling, phosphorylation-specific antibodies, ELISA, antibody arrays, flow cytometry, and phosphoproteomics. Phosphopeptide enrichment is generally required before mass spectrometric analysis to improve detection of low-abundance phosphorylation sites.

7 Protein Glycosylation

7.1 Major Types of Glycosylation

(1) N-Glycosylation

N-glycosylation mainly occurs on asparagine residues, is initiated in the endoplasmic reticulum, and is further processed in the Golgi apparatus, participating in protein folding, quality control, transport, and secretion.

(2) O-Glycosylation

O-glycosylation mainly occurs on serine or threonine residues, and its glycan structures and modification sites are highly diverse, participating in mucin formation, cellular signaling, and regulation of protein stability.

(3) C-Glycosylation

C-glycosylation mainly involves formation of a carbon-carbon bond between a glycan and a specific tryptophan residue. C-mannosylation is a common form and can affect protein folding, secretion, and receptor function.

7.2 Biological Functions of Glycosylation

Glycosylation is widely present in secreted and membrane proteins and can regulate protein folding, degradation, secretion, transport, and clearance while also participating in cell adhesion, cell recognition, receptor activation, endocytosis, and immune responses. Certain glycosylation modifications in the nucleus and cytoplasm have relatively simple structures but are highly dynamic .

7.3 Methods for Glycosylation Research

Glycosylation can be studied through lectin binding, glycosidase treatment, glycosyltransferase activity assays, chromatographic separation, and mass spectrometric analysis. Glycoproteomics generally uses lectin affinity, hydrophilic interaction, or chemical-labeling methods to enrich glycoproteins and glycopeptides, followed by mass spectrometry to identify glycan composition, glycosylation sites, and carrier proteins.

8 Protein Methylation

8.1 Lysine Methylation

The ε-amino group of the lysine side chain can undergo monomethylation, dimethylation, and trimethylation. Different degrees of methylation and modification sites can generate distinct protein-recognition interfaces and participate in heterochromatin formation, activation or repression of gene transcription, and DNA damage responses.

8.2 Arginine Methylation

Arginine can undergo monomethylation, asymmetric dimethylation, and symmetric dimethylation. Arginine methylation can regulate RNA binding, protein interactions, transcriptional regulation, signal transduction, and nucleocytoplasmic transport.

8.3 Methods for Methylation Research

Protein methylation can be detected using modification-specific antibodies, immunoblotting, immunofluorescence, immunoprecipitation, and mass spectrometric analysis. Histone methylation can also be studied using ChIP-qPCR or ChIP sequencing to analyze the distribution of specific modifications across chromatin regions and their relationships with gene transcription.

9 Protein Acetylation

9.1 Major Forms of Acetylation

Protein acetylation mainly includes N-terminal acetylation and lysine acetylation. N-terminal acetylation occurs at the protein N terminus and is generally relatively stable, whereas lysine acetylation is catalyzed by lysine acetyltransferases and can be removed by histone deacetylases or Sirtuin-family proteins.

9.2 Regulatory Roles of Acetylation

Lysine acetylation can neutralize the positive charge of lysine and alter protein binding to DNA, RNA, or other proteins. Acetylation participates in chromatin opening, gene expression, metabolic-enzyme activity, protein stability, nucleocytoplasmic transport, cell-cycle regulation, and cytoskeletal regulation.

9.3 Methods for Acetylation Research

Acetylation can be analyzed using pan-acetyllysine antibodies or site-specific antibodies for immunoblotting, immunofluorescence, and immunoprecipitation, as well as through acetyltransferase or deacetylase activity assays and mass spectrometric analysis. Acetylproteomics generally combines enrichment using anti-acetyllysine antibodies with LC-MS/MS identification of differentially modified sites.

10 Protein Lactylation

10.1 Metabolic Basis of Lactylation

Lactylation is a lysine acylation modification that connects lactate metabolism with regulation of protein function. Enhanced glycolysis, hypoxia, or lactate accumulation can alter intracellular levels of lactate and related metabolic intermediates, thereby affecting lysine lactylation of histone and nonhistone proteins .

10.2 Biological Functions of Lactylation

Histone lactylation can affect chromatin states and gene expression and participates in inflammatory regulation, immune-cell function, tumor immune evasion, tumor-cell migration, angiogenesis, treatment resistance, and extracellular-matrix remodeling. Lactylation levels are also affected by lactate production, lactate transport, oxygen supply, and cellular metabolic status.

10.3 Methods for Lactylation Research

Lactylation can be analyzed using pan-lysine-lactylation antibodies for immunoblotting, immunofluorescence, and immunoprecipitation. Liquid chromatography-tandem mass spectrometry can identify lactylated peptides and specific sites and can be used to compare changes in the lactylated proteome under different treatment conditions.

11 Experimental Design for Post-Translational Modification Research

11.1 Sample Processing and Preservation of Modifications

Protein extraction should be performed under low-temperature conditions, and lysis-buffer composition, sample-processing time, and centrifugation conditions should remain consistent among groups. For highly dynamic modifications, cellular reactions should be terminated rapidly and corresponding inhibitors should be added to prevent dephosphorylation, deubiquitination, or deacetylation during sample processing.

11.2 Modification Enrichment and Total-Protein Correction

Modification-omics requires not only comparison of modified-peptide abundance but also measurement of the total abundance of corresponding proteins. An increase in a target modification may result from an increased modification ratio per unit of protein or from an increase in total protein abundance. Therefore, modification signals should be corrected using total-proteomic data, target-protein immunoblotting, or immunoprecipitation results.

11.3 Site Identification and Functional Mutagenesis

Candidate sites identified by mass spectrometry should be screened using localization probability, replicate samples, fragment-ion coverage, and modified-peptide abundance. For functional validation, lysine, serine, threonine, or tyrosine can be replaced with residues that cannot accept the corresponding modification, followed by comparison of protein activity, stability, localization, and cellular phenotypes before and after mutation.

11.4 Combined Analysis of Multiple Modifications

The same protein may simultaneously undergo phosphorylation, ubiquitination, acetylation, methylation, or lactylation, and different modifications may exhibit cooperative, competitive, or sequential regulation. Combined analysis should integrate site proximity, temporal changes, modification-enzyme activity, and protein function and should avoid directly interpreting changes in a single modification as the final biological effect.

11.5 Common Problems in Result Interpretation

12 Products Related to Protein Post-Translational Modification Research

References

[1] Zhong Q, et al. Protein Posttranslational Modifications in Health and Diseases: Functions, Regulatory Mechanisms, and Therapeutic Implications. MedComm (2020). 2023 May 2;4(3):e261.

[2] Luo P, et al. The Role of SUMOylation in the Neurovascular Dysfunction After Acquired Brain Injury. Front Pharmacol. 2023 Mar 22;14:1125662.

[3] Reily C, et al. Glycosylation in Health and Disease. Nat Rev Nephrol. 2019 Jun;15(6):346-366.

[4] Izzo LT, et al. Histone Lactylation Links Metabolism and Gene Regulation. Nature. 2019 Oct;574(7779):492-493.

For more related articles, please see below:

[1] Phosphorylated and Sulfonated Peptides

[2] Glycosylation in Biopharmaceuticals

[3] Structural Coding of the Ubiquitin System and Mechanisms of Protein Homeostasis Regulation

[4] Ubiquitin–Proteasome System (UPS)

[5] SUMO Tag Grade Reagents