Protein Post-Translational Modifications

Translating Diversity to Discovery

Introduction

The field of proteomics focuses on studying the entire complement of proteins within a cell, tissue, or organism to unravel the intricate details of biological processes or disease states. Based on the “one gene = one protein” hypothesis, the process of translation generates approximately 20,000 canonical proteins. However, the presence of diverse protein isoforms or ‘proteoforms’ produced by alternative splicing, nucleotide polymorphisms and post-translational modifications (PTMs) significantly amplifies the coding flexibility of the transcriptome. PTMs in particular create a highly dynamic cellular environment through the reversible or irreversible addition of chemical moieties to specific amino acid residues of a protein. These modifications can alter the structure, function and localization of proteins, adding to the spatial and temporal control of protein function. While the exact number is continually expanding with ongoing research, it is estimated that proteins can be decorated with hundreds of distinct chemical modifications. These include phosphorylation, glycosylation, acetylation, methylation, ubiquitination, sumoylation, among many others. Aberrant expression of these PTM markers is associated with the development of various diseases, including cancer, neurodegenerative disorders, immune disorders, metabolic conditions, and cardiovascular diseases. Despite their significance, their comprehensive analysis has been hindered by available analytical techniques.

Phosphorylation

Protein phosphorylation is the most abundant protein PTM that affects most basic cellular processes. The majority of phosphorylation events occur on serine, threonine and tyrosine amino acid residues and are mediated by the opposing actions of protein kinases and phosphatases. The human genome includes ~568 kinases and 156 protein phosphatases that regulate phosphorylation events, and which may themselves be regulated by phosphorylation or de-phosphorylation.

Protein kinases phosphorylate proteins by the addition of the anionic phosphate group (PO₄). This increases the protein’s hydrophilicity, causing it to change conformation and influencing its interactions with other proteins. Up to 30% of all human proteins are modified by kinase activity. Phosphatases have the opposite function to kinases – hydrolytic removal of the phosphate group from the protein.

Major cellular processes regulated by phosphorylation include cell cycle progression, the DNA damage response, cell growth, differentiation, apoptosis, and many others.

Glycosylation

Protein glycosylation refers to the covalent attachment of glycans (carbohydrates, saccharides, or sugars) to proteins, which is the prevailing form of post-translational modification observed in excreted and extracellular membrane proteins. This process primarily occurs in the endoplasmic reticulum and Golgi apparatus of cells. Glycosylation can be broadly categorized into two types: N-linked glycosylation, where glycans bind to the amino groups of asparagine residues, and O-linked glycosylation, where glycans bind to the hydroxyl groups of serine and threonine residues.

Glycoconjugates typically consist of 17 different monosaccharides, which can be combined in diverse ways to create unique glycan structures. Enzymatic site preferences and the various configurations of glycosidic bonds (α or β conjugations) significantly contribute to the wide range of sugar linkages, leading to an astonishing potential for approximately 10¹² distinct branched glycan structures. As a result, the glycoproteome is greatly diversified.

Glycosylation plays a vital role in various biological processes, such as facilitating cell adhesion to the extracellular matrix and mediating protein-ligand interactions within cells. Changes in glycan composition are directly linked to many diseases, including congenital disorders, immune responses, cancer, autoimmune diseases, and chronic inflammatory diseases.

Ubiquitination

Ubiquitin is a highly conserved, 76-amino acid protein widely present in eukaryotic cells. The process of ubiquitination involves covalently attaching ubiquitin to lysine residues of target proteins. This process is facilitated by three types of enzymes: ubiquitin activating enzymes - E1, ubiquitin conjugating enzymes - E2, and ubiquitin ligases - E3. Ubiquitination acts as a versatile signaling mark, modifying substrates through mono-ubiquitination (addition of a single ubiquitin molecule) or poly-ubiquitination (conjugation of ubiquitin to preceding moieties). Mono-ubiquitination functions as a signaling mark for DNA repair, transcription regulation, cell signaling and the immune response, while poly-ubiquitination directs substrate proteins for degradation via the 26S proteasome — a process responsible for degradation of ~80% of intracellular proteins. Dysregulation of the ubiquitin system is linked to neurodegenerative disease such as Alzheimer’s, Parkinson’s, and Huntington’s disease.

Acetylation

Protein acetylation is a dynamic process that involves the addition of acetyl groups to lysine residues in proteins. Acetylation predominantly occurs on the N-terminal tails of histone proteins, which are components of chromatin—the complex of DNA and proteins that form chromosomes. Histone acetylation is associated with relaxed chromatin structure, making the underlying DNA more accessible to transcriptional machinery. This “open” chromatin state allows for increased gene transcription, leading to enhanced gene expression. By influencing gene expression, acetylation plays a crucial role in diverse cellular processes, including cell cycle regulation, DNA repair, differentiation, and apoptosis. Its influence extends beyond histones; numerous non-histone proteins, including transcription factors, chaperones, and enzymes, can also undergo acetylation, affecting their activities, interactions, and stability. Importantly, acetylation is a reversible modification, as acetyl groups can be added by acetyltransferases and removed by deacetylases. Maintaining the proper balance between acetylation and deacetylation is essential for optimal cellular function, as dysregulation of protein acetylation has been associated with various diseases, including cancer, neurodegenerative disorders, and metabolic disorders.

Palmitoylation

Protein palmitoylation is a reversible lipid modification that involves the addition of a palmitate molecule (a 16-carbon saturated fatty acid) to cysteine residues in proteins. This process, known as S-palmitoylation, plays a crucial role in the regulation of protein localization, stability, and function. The enzyme palmitoyl acyltransferases catalyze the transfer of palmitate to target proteins in the endoplasmic reticulum and Golgi apparatus. Protein palmitoylation contributes to the membrane association of proteins, influencing their trafficking, subcellular localization, and interactions with other proteins and lipids. It is particularly important for proteins involved in signal transduction, synaptic transmission, and membrane trafficking. Palmitoylation can modulate protein-protein interactions, membrane microdomain partitioning, and stability, thus impacting various cellular processes and pathways.

Sumoylation

Sumoylation is a PTM where a small protein called Small Ubiquitin-like Modifier (SUMO) is covalently attached to specific lysine residues in target proteins. Sumoylation can affect protein activity, stability, subcellular localization, and protein-protein interactions. It plays a significant role in diverse cellular processes such as transcriptional regulation, DNA repair, nuclear transport, and protein degradation. By modifying target proteins, sumoylation influences their function and interactions with other molecules, ultimately impacting cellular responses. Dysregulation of sumoylation have been associated with various diseases, including cancer, neurodegenerative disorders, and viral infections.

Methylation

Protein methylation is a reversible post-translational modification that involves the addition of a methyl group (-CH3) to specific amino acid residues, predominantly lysine and arginine, in proteins. This process catalyzed by protein methyltransferases, can be reversed by protein demethylases, enzymes that remove methyl groups from modified proteins. Protein methylation impacts numerous cellular processes, including transcriptional regulation, chromatin remodeling, DNA repair, and signal transduction. It can serve as a molecular switch, modulating protein interactions and influencing protein stability and function.

Oxidation

Protein oxidation refers to the process of oxidative damage to proteins within cells and tissues. It occurs when proteins are exposed to reactive oxygen species (ROS), or reactive nitrogen species generated during normal cellular metabolism (respiration, immune responses and redox signaling) or in response to various environmental stressors (radiation, pollution and toxins). ROS are highly reactive molecules that contain oxygen atoms with unpaired electrons. Common ROS includes the superoxide anion (O2^-), hydrogen peroxide (H₂O₂), and hydroxyl radical (OH), while nitric oxide (NO) is a reactive nitrogen species. Proteins are susceptible to oxidation due to their abundant amino acid residues with side chains that can react with ROS. The amino acid residues most susceptible to oxidation include cysteine, methionine, histidine, tyrosine, and tryptophan. Protein oxidation can result in various modifications, including the formation of carbonyl groups.

Protein oxidation can disrupt the three-dimensional structure of proteins, alter their enzymatic activity, affect protein-protein interactions, and impair their folding and degradation. These changes can lead to loss of protein function and may contribute to cellular dysfunction and aging. While some level of oxidation is necessary for certain cellular processes (cellular metabolism, immune responses, and redox signaling), excessive or uncontrolled oxidative stress can damage cellular components, including lipids, proteins, and DNA. This can lead to a range of health issues, including neurodegenerative diseases, cardiovascular diseases, and cancer.

Nitrosylation

Nitrosylation is a post-translational modification of proteins in which a NO group is covalently attached to a specific amino acid residue, typically cysteine, resulting in the formation of an S-nitrosothiol — SNO group. NO is a highly reactive free radical that can be produced by various cellular processes and enzymes, including nitric oxide synthases or during the metabolism of nitrite and nitrate. NO has a short half-life and can readily diffuse through cell membranes, allowing it to interact with nearby biomolecules, including proteins. Under conditions of increased NO production or oxidative stress, NO can react with cysteine residues in proteins, leading to the formation of S-nitrosothiols. This process can occur spontaneously, without the requirement for specific enzymes or catalysis. It’s worth noting that while spontaneous protein nitrosylation can occur, it is also regulated by various enzymes called nitrosylases and denitrosylases, which add or remove NO groups from proteins, respectively. These enzymatic processes help maintain the balance of protein nitrosylation and denitrosylation in cells. This delicate equilibrium ensures that protein S-nitrosylation remains tightly controlled, contributing to overall cellular homeostasis and physiological functions.

A recent advancement in the study of nitrosylation is the biotin-switch method. This is a biochemical technique used to identify and quantify S-nitrosylated proteins in biological samples. It involves blocking free thiol groups, reducing S-nitrosylated cysteines back to their thiol form, and selectively biotinylating the now-reduced free thiols. Biotinylated proteins can then be identified using various techniques such as fluorescent labeling and mass spectrometry.

Detecting Protein PTMs: Methods and Techniques

PTMs play a crucial role in altering a protein’s conformation, localization, stability, activity, and interactions with other proteins, ultimately influencing fundamental biological processes. Thus, abnormal PTMs can disrupt these functions, contributing to the development of disease. Consequently, the reliable and sensitive identification and quantification of PTMs is crucial to understanding disease mechanisms. Although proteomic techniques have made significant advances, unraveling the roles and implications of many PTMs remains a formidable task. In the subsequent section, we shed light on the commonly employed techniques embraced by researchers and clinicians for the detection and quantification of PTMs.

Mass Spectrometry (MS)

Mass spectrometry is a powerful technique for PTM analysis. It allows for the identification and quantification of modified peptides in complex protein mixtures. Various MS methods, such as liquid chromatography-mass spectrometry (LC-MS) or tandem mass spectrometry (MS/MS), can be employed to identify and localize PTMs. Two mass spectrometric approaches are commonly used in clinical settings: electro-spray ionization (ESI) MS suitable for liquid samples like plasma or urine, and matrix-assisted laser desorption/ionization (MALDI) MS which is relevant particularly for tissue biopsies.

There are three main approaches in mass spectrometry PTM detection: top-down, middle-down, and bottom-up.

The major steps involved in mass spectrometry PTM detection, regardless of the approach (top-down, middle-down, or bottom-up), can be summarized as follows:

Antibody-based methods

Antibody-based techniques (immunoassays) rely on the use of specific antibodies that recognize and bind to the modified form of the protein or the PTM of interest. Below are some common antibody-based methods for PTM detection.

While the above antibody-based techniques are useful for detection of a few PTMs at a time, large-scale highthroughput identification of PTMs can be achieved through the use of protein microarrays or antibody arrays. Unlike traditional labor-intensive techniques like mass spectrometry or western blotting, arrays offer a high-throughput and multiplexed approach to simultaneously detect and quantify multiple PTMs in a single experiment.

Protein Microarrays

These are high-throughput platforms that enable the simultaneous analysis of a large number of PTMs. They allow researchers to screen for specific modifications across a diverse set of proteins. The process involves immobilizing proteins or peptides onto a solid support, such as a glass slide or a nitrocellulose membrane, creating an array of distinct spots. Each spot represents a unique protein or peptide. specific PTM-specific antibodies or PTM-binding domains are used as probes. These probes selectively bind to the target PTMs on the immobilized proteins or peptides. The bound probes can be detected and quantified using fluorescence, chemiluminescence, or other detection methods.

Antibody Arrays

Antibody arrays are a variation of protein microarrays that utilize an arrayed collection of specific antibodies immobilized on a solid support, such as a glass slide or a nitrocellulose membrane. The antibodies are carefully selected to target and recognize specific PTMs on proteins of interest. While the selection, validation, and optimization of the antibody panel is laborious and costly, the finished array is an easy-to-use tool for the researcher, allowing rapid and precise data output for PTM screening. The array spot signals are captured typically by laser fluorescent scanner or chemiluminescent imager; the raw data can then be mapped quickly to its corresponding analyte using an extraction grid.

The workflow for PTM detection using antibody arrays typically involves the following steps.

• Sample preparation: Extract and enrich proteins for PTMs.
• Incubation: Bind PTM-specific antibodies to proteins.
• Washing: Remove non-specific bindings.
• Detection: Detect modified proteins using signals.
• Data analysis: Analyze PTM levels and compare conditions.

Protein PTMs can be detected using a variety of technical variations of antibody arrays. One approach involves directly spotting antibodies specific to the PTM of interest onto a solid support. When samples containing proteins or peptides are applied to the array, the target PTMs in the samples bind to the immobilized antibodies. Detection methods are then used to visualize and quantify the bound PTMs.

Another technique involves spotting antibodies specific to target proteins onto a solid support, and then incubating samples to allow proteins to bind to the arrayed antibodies. PTMs are subsequently detected using PTM-specific labeled detection antibodies, which are visualized using fluorescent label chemistry. This sandwich antibody technique ensures highly specific PTM detection.

Besides these methods, other approaches like the biotin-switch (mentioned earlier) and acyl-biotinyl exchange methods, derivatization (applying chemical modifications to specific PTMs for enhanced detection), and chemical blocking of unmodified residues (selectively removing unmodified residues to focus on PTM analysis) offer additional detection strategies for PTM screening.

Leveraging the cutting-edge techniques mentioned above along with meticulously validated antibodies, RayBiotech has developed a first-of-its-kind solution for high-throughput PTM detection across 8000 target analytes. As the sole provider of this unparalleled technology and an expanding pipeline of tools, we are committed to facilitating proteomic discoveries through PTM screening. In addition, discover the convenience of PTM detection at the benchtop with our colorimetric PTM detection kits and modification kits for western blotting. Our comprehensive range includes acylation, nitrosylation, carbonyl content kits, lipid peroxidation kits, and more, for easy and efficient screening of PTMs.

Check out the table below to explore our wide selection of currently available PTM arrays and detection kits.

Presented below are examples of experimental data highlighting the application of RayBiotech’s PTM detection antibody arrays.

Figure 1. Protein S-Nitrosylation Antibody Array

Detection of Protein S-Nitrosylation using the RayBio^® Human Receptor Tyrosine Kinase Antibody Array (cat no.  AAH-PRTK-G1), which detects 71 human receptor tyrosine kinases. The Biotin-Switch technique was used to covalently attach biotin to previously S-nitrosylated cysteine residues. Left – Untreated Huvec/A431 cell lysates.
Right – Huvec/A431 cell lysates treated with 200 µM S-Nitrosoglutathione for 30 minutes at 37°C (to induce Nitrosylation) revealed an increased fluorescent signal intensity of target proteins.

Detection of protein acylation (palmitoylation) using the RayBio^® Human Protein S-Acylation Array G2 [cat no.  AAH-ACYL-G2] which detects 493 human proteins. A modified biotin switch technique, known as the acyl-biotinyl exchange method, was used to identify acylated cysteines. Left: untreated cells. Right: HEK293 cell lysates treated with 100 µM palmitoyl-CoA for 1 hour at 37°C showing increased fluorescence intensity.

Detection of protein oxidation using the RayBio^® Human Protein Oxidation Array G1 [cat no. AAH-OXI-G1] that detects 507 human proteins. Oxidation was detected using ‘Biotin Probes’ which react with carbonyl groups in proteins to form unstable Schiff bases, which are then reduced to more stable amines that can be fluorescently visualized. Left: Untreated Hela cells, Middle: Hela cell lysates treated with 50 mM sodium borohydride for 30 minutes to reduce oxidation, showing decreased fluorescence signal and Right: Hela cells treated with 1 mM hydrogen peroxide for 15 minutes to induce oxidation, showing increased fluorescence intensity.