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Overview: N-terminus and C-terminus in Protein

Proteins are composed of linear chains of amino acids, with each protein having two distinct ends: the N-terminus, featuring a free amine group, and the C-terminus, having a free carboxyl group. These termini are essential for the protein's function, stability, and interaction with other molecules. The analysis of these termini is crucial in protein engineering, drug development, and quality control of biopharmaceuticals.

Significance of N-terminal and C-terminal Analysis

Research on Protein Physiological Functions

Analyzing the N-terminus and C-terminus of proteins is crucial due to the significant roles these terminal regions play in protein functionality, localization, and stability. The diversity of protein forms generated from a single gene through mechanisms such as co- or post-translational modifications, alternative splicing, and alternative translation initiation makes terminal analysis indispensable for understanding protein biology.

Verification of Protein Terminal Modifications

Precise chemical modification of proteins can improve their physicochemical properties and endow them with new physiological functions, such as extending protein half-life and regulating protein interactions. The N-terminus of a protein or peptide drug is crucial as it determines the site and efficacy of the drug's action. Changes at the N-terminus, including alterations and modifications of the N-terminal amino acids during the drug manufacturing process or storage, are common. In contrast to the development of modification techniques for protein side chains and N-termini, precise strategies for C-terminal modifications are still significantly lacking. During the modification process of protein N-termini or C-termini, it is essential to monitor and verify their sequences to obtain correctly modified samples.

Terminomics Analysis (High Throughput)

With the rise of proteogenomics, re-annotating genomes using proteomics data has become a research hotspot. Employing proteomics methods for large-scale identification and analysis of protein termini helps verify and correct annotated genes and even discover new genes. Over the past 20 years, mass spectrometry technology has undergone rapid development, leading to more in-depth studies on the proteomics of protein termini. The establishment and improvement of various terminal enrichment techniques and methods have gradually matured large-scale sequencing of protein termini.

Quality control of biopharmaceutical products

Analyzing the N- and C-termini is vital across a broad spectrum of biological products, including proteins, antibodies, vaccines, polypeptides, and recombinant collagen. Such analysis ensures the structural integrity and biological activity of these products. For instance, in monoclonal antibody (mAb) quality control, identifying and quantifying terminal amino acid sequences in light and heavy chains is crucial. Multiple terminal sequences must be accurately determined to ensure product consistency and efficacy.

Methods of N-terminal and C-terminal Analysis

Advances in proteomics have introduced diverse technologies for terminal analysis, each offering unique benefits and insights.

N-terminal Sequencing

Currently, there are two primary methods for N-terminal sequencing of proteins.

The first method is Edman degradation, which sequentially cleaves and analyzes the limited number of amino acids from the N-terminus. This method determines the N-terminal amino acid sequence by comparing the retention times of amino acid standards.

The second method is mass spectrometry, where the protein is denatured, reduced, and enzymatically digested into peptides of varying sizes. These peptides are then separated by liquid chromatography and introduced into a high-resolution mass spectrometer at different time intervals. The m/z values of the peptides are analyzed and compared against a proposed sequence.

Depending on the conditions and the amount of amino acid data required, these two methods are often combined or chosen based on comprehensive considerations.

Edman degradation

Principle

Edman degradation is a process for determining the amino acid sequence of a peptide starting from the free N-terminus. Under alkaline conditions, phenylisothiocyanate (PITC) reacts with the N-terminal amino group of the protein or peptide to form a phenylthiocarbamoyl (PTC) derivative. This derivative is then treated with acid, causing cyclization and selective cleavage of the N-terminal residue, producing a phenylthiohydantoin (PTH) derivative of the N-terminal amino acid. The cyclic peptide then enters the next cycle. The PTH derivative is extracted with an organic solvent, and under acidic conditions, it reacts to form a stable phenylthiohydantoin (PTH) derivative. This PTH derivative is analyzed by HPLC to identify the amino acid. Each cycle provides the information of one amino acid.

Procedure

  • Sample Preparation: Before protein sequencing, the protein sample is separated by SDS-PAGE to ensure purity. The protein on the SDS-PAGE is then transferred to a PVDF membrane. After staining, the protein bands are cut out and analyzed using a protein sequencer.
  • Analysis: Edman degradation sequencing involves cyclic reactions that sequentially identify amino acids from the N-terminus of the protein. Each Edman sequencing reaction consists of three steps: first, PITC reacts with the free amino group at the N-terminus under alkaline conditions; second, the N-terminal residue is cleaved in an acidic solution; third, the PTC derivative is converted into a more stable PTH derivative, which is then analyzed by HPLC to determine the type of amino acid based on its retention time.

Combination of Edman degradation with LC-MS workflowFigure 1. Combination of Edman degradation with LC-MS workflow. (Anna A. et al., 2013)

Instrumentation

Based on the steps of Edman amino acid sequencing, instrument companies have developed protein sequencers, which include two main modules: a sample processing module and a liquid chromatography module. The sample processing module automates coupling, cleavage, extraction, and conversion, while the liquid chromatography module automatically analyzes the obtained PTH-amino acids, identifying the amino acid types.

Advantages

  • High Reaction Yield and Recovery: PITC reacts efficiently with all amino acid residues, resulting in high yield and recovery rates. Consequently, the formation of by-products is minimal, allowing for accurate identification through chromatography.
  • Fast Reaction Time: The Edman degradation process is relatively quick. For most amino acid residues, the coupling reaction requires only about 30 minutes, and the cleavage reaction can be completed in just 5 minutes.
  • Retention of Intact Peptide Chains: After the reaction, the peptide chain remains intact, enabling repeated Edman degradation cycles to determine newly exposed N-terminal amino acids. This allows for sequential and thorough analysis of the N-terminal sequence.

Disadvantages

  • Limitations with Modified or Blocked N-Termini: Peptides that have undergone chemical modifications or have blocked N-termini cannot be effectively analyzed using Edman degradation.
  • Low Throughput: Edman degradation is classified as a low-throughput method. While it is reliable for sequencing, it can only analyze approximately 30 amino acids in a single run when sample quantities are adequate.

Mass Spectrometry (MS)

Principle

The most widely used method for protein sequencing currently is mass spectrometry (MS). MS-based protein sequencing strategies are broadly categorized into two main approaches: Top-Down and Bottom-Up.

Top-Down Strategy: This approach analyzes intact proteins directly using liquid chromatography-mass spectrometry (LC-MS) without prior degradation. The protein's sequence is determined by identifying fragment ions in the mass spectra.

Bottom-Up Strategy: In this approach, proteins are first hydrolyzed into peptides. These peptides are then analyzed using LC-MS, where they are sequenced de novo and their sequences are pieced together to reconstruct the complete protein sequence.

Procedure

Top-Down Approach

  • Separation and Ionization: Intact proteins are separated from complex biological samples using reverse-phase liquid chromatography. The proteins are ionized using electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI).
  • Fragmentation: The resulting ions are fragmented through collision-induced dissociation (CID), high-energy collision dissociation (HCD), electron capture dissociation (ECD), or electron transfer dissociation (ETD).
  • MS Analysis: The fragments are analyzed by tandem mass spectrometry (MS/MS).

Bottom-Up Approach

  • Digestion: Protein mixtures are digested into peptide mixtures. The peptide mixtures are separated by chromatography and ionized.
  • MS Analysis: The ionized peptides are analyzed by tandem mass spectrometry (MS/MS) to generate peptide fingerprints, which are used for peptide identification.

MS/MS analysis of the N-terminal for the light chain for mAb1

Figure 2. CID MS/MS spectra of the N-terminal tryptic peptide of the light chain for mAb1. (Malgorzata Monika et al., 2019)

Instrumentation

High-performance liquid chromatography-tandem mass spectrometry (LC-MS) consists of two main components:

  • High-Performance Liquid Chromatography (HPLC): Used for the separation of proteins.
  • Tandem Mass Spectrometry (MS/MS): Used for ionizing proteins and analyzing their fragments.

Advantages

  • N-Terminal Sequencing with High-Resolution Mass Spectrometry: This method allows for the analysis of N-terminal blocking and post-translational modifications (PTMs) through high-resolution mass spectrometry, determining the protein's N-terminal starting point.
  • Complex Protein Analysis: By analyzing the labeling positions (such as N-terminal dimethylation labeling), it is possible to determine the N-terminal starting points of multiple proteins in a complex protein sample.
  • Identification of Non-Standard Amino Acids: Mass spectrometry can identify non-standard amino acids that are difficult to determine using traditional chemical methods due to the lack of reference standards.

Disadvantages

  • Database Dependence: Mass spectrometry identification often relies on database comparison for protein sequence analysis. Therefore, proteins not included in the database cannot be accurately analyzed by mass spectrometry.
  • Incomplete Sequence Coverage: Under current conditions, mass spectrometry rarely provides 100% sequence coverage of peptides. Some peptide fragments may be missed.
  • Interference Issues: The accuracy of analysis can be affected by protein contamination, isotopic peak interference, and incomplete peptide fragmentation.

C-terminal Sequencing

Carboxypeptidase Digestion

Principle

Carboxypeptidases are exopeptidases that specifically hydrolyze peptide bonds at the C-terminus of proteins, releasing amino acids one at a time. The number and type of amino acids released vary with reaction time. By analyzing the amount of amino acids released over time, the sequence of amino acids at the C-terminus can be determined.

Procedure

  • Enzymatic Digestion: The enzyme cleaves amino acids sequentially from the C-terminus, releasing free amino acids over time.
  • Analysis: The identity of the peptide sequence can subsequently be deduced by HPLC-based detection of released residues (considering the release order) or by analyzing the truncated peptides using MALDI-TOF-MS.

Instrumentation

The analysis method depends on the approach used:

  • High-Performance Liquid Chromatography (HPLC): Used for direct detection of amino acids released by hydrolysis.
  • Mass Spectrometry (MS): Analyze the difference in molecular weight between the original protein or peptide and the truncated fragments to determine the molecular weight of the released amino acids.

Advantages

  • High Molecular Weight Protein Analysis: By combining the chemical cleavage of proteins with cyanogen bromide and the specific degradation of the C-terminus using carboxypeptidases, it is possible to use mass spectrometry to detect the peptide fragments obtained at different digestion times.
  • Enhanced Precision and Sensitivity: The catalytic hydrolysis conditions of carboxypeptidase can be optimized by collecting data at different digestion time points, thereby improving the accuracy and sensitivity of the analysis.

Disadvantages

  • High Cost of Optimization: Since the cleavage rate of carboxypeptidase largely depends on the peptide sequence, this method usually requires careful optimization of reaction conditions, making it labor-intensive and requiring relatively large amounts of sample.

Mass Spectrometry (MS)

Principle

MS-based methods for C-terminal sequencing of proteins utilize two main approaches: top-down and bottom-up. The bottom-up approach is widely used due to its practical advantages, including higher sensitivity for low-abundance proteins, easier handling of complex protein mixtures, and compatibility with existing proteomic workflows. In the bottom-up approach, proteins are enzymatically digested into peptides, which are then analyzed by mass spectrometry (MS). It can further be categorized into two main strategies:

Labeling Strategies

Labeling strategies involve chemically modifying peptides to introduce tags or labels that aid in their detection and sequencing by MS. These strategies enhance the ionization efficiency and distinguish C-terminal peptides from other peptide fragments in the mixture. Examples include:

  • 18O-Labeling: Incorporation of 18O atoms at the C-terminal carboxyl group during protease digestion, allowing for differential mass tagging.
  • Isotopic Labeling: Introduction of stable isotopes (e.g., 13C or 15N) into peptides, enabling quantitative analysis and identification of C-terminal peptides.

Enrichment strategies

Enrichment strategies focus on isolating C-terminal peptides from complex peptide mixtures before MS analysis. This selective enrichment improves sensitivity and specificity in identifying C-terminal sequences. Common enrichment methods include:

  • Positive Selection: Direct targeting and isolation of C-terminal peptides using affinity tags or antibodies specific to C-terminal sequences.
  • Negative Selection: Depletion of non-C-terminal peptides, leaving enriched C-terminal peptides for subsequent MS analysis.

Procedure

The methods encompassed within Labeling Strategies and Enrichment Strategies are highly diverse. Here, two straightforward approaches are outlined respectively:

Labeling Strategies

  • Digestion: Enzymatic digestion of proteins is conducted in a buffer solution containing H218O, leading to the incorporation of one or two oxygen-18 atoms by the protease at the carboxyl moiety of the cleaved peptide bond. This process does not modify the carboxyl group of the original C-terminal peptide.
  • MS Analysis: In MS1 analysis, C-terminal peptides appear as singular peaks, whereas N-terminal and internal peptides exhibit an isotope envelope reflecting the incorporation of 18O atoms (resulting in a 2 or 4 Da mass shift).

Schematic representation of methods for C-terminal labeling

Figure 3. Schematic representation of methods for C-terminal labeling. (A) Protease-assisted 18O-labeling. (B) Method to differentiate C-terminal peptides in cyanogen bromide digests. (C) Isotopic arginine labeling based on the oxazolone chemistry. (Sebastian et al., 2015)

Enrichment strategies

The COFRADIC technology exploits peptide chromatography to specifically isolate peptides of interest from complex peptide mixtures.

  • Digestion: After acylating all primary amines, the proteome is subjected to trypsin digestion.
  • Separation: Strong cation exchange (SCX) fractionation is carried out under acidic conditions (pH 3.0) on the tryptic digest. An additional step involves chemical derivatization of primary amines present in C-terminal peptides using an N-hydroxysuccinimide ester of butyrate. This step is performed between two consecutive RP-HPLC separations to further segregate C-terminal peptides from N-terminal peptides.
  • MS Analysis: The enriched C-terminal peptides are analyzed using LC-MS/MS instrumentation.

Schematic representation of C-terminal enrichment strategies

Figure 4. Schematic representation of C-terminal enrichment strategies. C-terminal COFRADIC (A) and C-TAILS (B) represent negative selection strategies for C-terminal peptide enrichment. On the other hand, ProC-TEL (C) applies a positive selection to isolate C-termini. (Sebastian et al., 2015)

Instrumentation

MS-based C-terminal sequencing requires advanced instrumentation:

  • High-resolution Mass Spectrometers: Such as Time-of-Flight (TOF) instruments, for accurate mass measurement of peptides.
  • Liquid Chromatography (LC): Coupled with MS (LC-MS), for peptide separation and enhanced resolution.

Advantages

  • Enhanced Recognition Efficiency: C-terminal peptides typically represent only a small fraction of all peptides generated from protein digestion. Methods involving labeling or enrichment improve the efficiency of identifying C-terminal peptides.
  • Improved Sensitivity and Accuracy: Mass spectrometry methods offer higher sensitivity and accuracy and are more cost-effective compared to enzymatic digestion approaches.

Disadvantages

  • Incomplete Sequence Coverage: Under current conditions, mass spectrometry rarely provides 100% sequence coverage of peptides. Some peptide fragments may be missed.

Conclusion

Understanding the N-terminus and C-terminus is fundamental in the field of protein biochemistry. At Creative Proteomics, we leverage this knowledge to explore innovative solutions in biotechnology, enhancing protein function, stability, and interaction capabilities. The distinct roles and structural characteristics of these termini underscore their importance in protein biology, providing a foundation for advanced research and application in various scientific and industrial domains.

References

  1. Lobas, Anna A. "Combination of Edman Degradation of Peptides with Liquid Chromatography/Mass Spectrometry Workflow for Peptide Identification in Bottom-up Proteomics." (2013).
  2. Vecchi, Malgorzata Monika. "Identification and Sequencing of N-Terminal Peptides in Proteins by LC-Fluorescence-MS/MS: An Approach to Replacement of the Edman Degradation." (2019).
  3. Tanco, Sebastian. "C-Terminomics: Targeted Analysis of Natural and Posttranslationally Modified Protein and Peptide C-Termini." (2015).
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Basic Structure of Antibodies

Antibodies exhibit a characteristic Y-shaped structure composed of four polypeptide chains: two identical heavy (H) chains and two identical light (L) chains. This structural arrangement is essential for their diverse functions in immune defense.

Composition of Antibodies

Heavy Chains (H Chains):

  • Structure: Each antibody molecule contains two heavy chains, which are longer and more complex than light chains. Heavy chains are encoded by gene segments known as V (variable), D (diversity), and J (joining), allowing for significant diversity in antibody structure and function.
  • Domains: Heavy chains consist of several domains, including a variable domain (VH) at the N-terminus and one or more constant domains (CH1, CH2, CH3, and sometimes CH4) towards the C-terminus. The VH domain is crucial for antigen binding and recognition due to its variability, determined by the V, D, and J gene segments.

Light Chains (L Chains):

  • Structure: Antibodies also contain two light chains, which are shorter and less complex than heavy chains. Light chains are encoded by gene segments V and J, similar to the V and J segments of heavy chains.
  • Domains: Light chains possess a variable domain (VL) and a constant domain (CL). The VL domain contains three hypervariable regions, also known as complementarity-determining regions (CDRs), which directly interact with antigens.

Y-Shaped Structure

The combination of heavy and light chains gives rise to the characteristic Y-shaped structure of antibodies, which is crucial for their function. The Y shape consists of two identical antigen-binding fragments (Fab) at the tips of the Y and one constant fragment (Fc) at the base of the Y.

  • Fab Fragments: Each Fab fragment consists of one variable domain from a heavy chain (VH) and one variable domain from a light chain (VL), forming the antigen-binding site. The specificity of antibody-antigen interactions is primarily determined by the unique combination of VH and VL domains.
  • Fc Fragment: The Fc fragment is composed of constant domains from the heavy chains (CH2 and CH3). It determines the antibody's class (e.g., IgG, IgA, IgM) and mediates interactions with other components of the immune system, such as complement proteins and Fc receptors on immune cells.

Disulfide Bonds and Stability

Antibodies are stabilized by disulfide bonds, covalent linkages formed between cysteine residues within and between the heavy and light chains. These bonds contribute to the structural integrity of antibodies, ensuring they remain functional under various physiological conditions.

Flexibility and Functionality

The hinge region between the Fab and Fc fragments provides flexibility to the antibody molecule, allowing each Fab fragment to move independently. This flexibility is essential for antibodies to bind to antigens with different spatial orientations and sizes, enhancing their efficacy in recognizing and neutralizing diverse pathogens.

Diagram of Antibody Structure

Below is a simplified diagram illustrating the basic structure of an antibody:

A schematic representation of the antibody structure

A schematic representation of the antibody structure (Golpour ET AL., 2021).

Structure of Heavy Chains

Gene Segments and Diversity of Heavy Chains

V (Variable), D (Diversity), and J (Joining) Segments: Heavy chains are encoded by genes that undergo somatic recombination of these segments during B cell development. This process generates extensive diversity in the variable regions of antibodies, enabling recognition of a wide range of antigens.

Types of Heavy Chains: Humans have five types of heavy chains, each associated with a specific class of antibody (Ig):

  • a (IgA), d (IgD), e (IgE), ? (IgG), and µ (IgM): These classes differ in their constant region sequences, which dictate their biological functions, such as opsonization, neutralization, and complement activation.

Structural Domains of Heavy Chains

Variable Region (VH): Located at the N-terminal end of each heavy chain, the VH domain is highly variable due to differences in the V, D, and J gene segments. It contains three complementarity-determining regions (CDRs) that directly interact with antigens, determining the specificity of antibody binding.

Constant Regions (CH1, CH2, CH3, and sometimes CH4): Found towards the C-terminal end of heavy chains, these constant domains exhibit less variability across antibody classes but are crucial for effector functions and antibody stability.

Structural Features and Flexibility

Hinge Region:

Located between the Fab and Fc fragments of antibodies, the hinge region of heavy chains provides flexibility and mobility to the antigen-binding arms. This flexibility allows antibodies to engage antigens at various angles and orientations, enhancing their effectiveness in immune defense.

Glycosylation:

Heavy chains, like many glycoproteins, undergo glycosylation, where carbohydrate molecules are attached to specific amino acid residues. These glycans can modulate antibody stability, solubility, and interactions with other immune components, influencing overall immune response dynamics.

Function and Importance of Heavy Chains

Antigen Binding:

The VH domain of heavy chains, along with the variable domain (VL) of light chains, forms the antigen-binding site of antibodies. This region contains three hypervariable loops, known as complementarity-determining regions (CDRs), which directly interact with antigens.

CDRs in the VH domain confer specificity to the antibody by recognizing and binding to unique epitopes on antigens, such as proteins on pathogens or abnormal cells.

Heavy chains contribute significantly to the affinity and specificity of antibody-antigen interactions. Through somatic recombination of V, D, and J gene segments, heavy chains generate a diverse repertoire of VH domains that can recognize a broad range of antigens with varying affinities.

Effector Functions:

Class Switching: Heavy chains determine the class or isotype of antibodies (e.g., IgG, IgA, IgM) through a process called class switching. This process occurs after initial antigen exposure and is influenced by cytokines and other signals from immune cells. Different antibody classes have distinct effector functions mediated by their constant regions, such as opsonization (IgG), mucosal immunity (IgA), and allergic responses (IgE).

Effector Mechanisms: The constant regions (CH2 and CH3) of heavy chains interact with complement proteins and Fc receptors on immune cells, initiating effector mechanisms that eliminate pathogens or infected cells. For example, IgG antibodies can activate the complement cascade, leading to pathogen lysis, or engage Fc receptors on phagocytes to facilitate pathogen clearance through phagocytosis.

Structural Stability:

The constant domains of heavy chains, particularly CH2 and CH3, contribute to the overall stability and structural integrity of antibodies. Disulfide bonds between cysteine residues within these domains reinforce the antibody's tertiary structure, ensuring functionality in diverse physiological environments.

Detailed Structure of Light Chains

Gene Segments and Diversity of Light Chains

V (Variable) and J (Joining) Segments: Light chains are encoded by genes that undergo somatic recombination of these segments during B cell development. This process generates diversity in the variable regions of antibodies, enabling them to recognize a wide range of antigens.

Types of Light Chains: There are two main types of light chains found in antibodies:

  • Kappa (?) Chains: The majority of antibodies (around 60-70%) in humans have kappa light chains.
  • Lambda (?) Chains: The remaining antibodies (around 30-40%) have lambda light chains.

Both kappa and lambda light chains perform similar functions in antigen recognition and binding, but their genetic origins and distributions differ.

Structural Domains of Light Chains

Variable Region (VL): Located at the N-terminal end of each light chain, the VL domain contains three hypervariable regions, also known as complementarity-determining regions (CDRs). These CDRs directly interact with antigens, determining the specificity and affinity of antibody binding.

Constant Region (CL): The CL domain is found towards the C-terminal end of light chains and provides structural stability to the light chain.

Comparative Analysis of Kappa and Lambda Chains

Distribution and Functionality:

  • Kappa (?) chains are more prevalent in antibodies produced by B cells, accounting for approximately 60-70% of total antibodies in humans.
  • Lambda (?) chains are less common, comprising the remaining 30-40% of antibodies.
  • Despite this difference in prevalence, both types of light chains contribute equally to antibody function and diversity in antigen recognition.

Genetic Diversity:

The genetic mechanisms that govern the expression of kappa and lambda light chains provide additional diversity in antibody repertoire, enabling the immune system to respond effectively to a wide array of pathogens and antigens.

Function and Importance of Light Chains

Antigen Binding:

The VL domain of light chains contains three hypervariable loops, or complementarity-determining regions (CDRs), which directly interact with antigens. CDRs in the VL domain, in conjunction with CDRs in the VH domain of heavy chains, determine the specificity and affinity of antibody-antigen interactions. Through somatic recombination of V and J gene segments, light chains generate a diverse repertoire of VL domains that can recognize a broad range of antigens with high specificity.

Stability and Flexibility:

Light chains contribute to the overall stability and flexibility of the antibody molecule. The interaction between light and heavy chains allows antibodies to adopt different conformations, optimizing antigen recognition and binding.

Pairing with Heavy Chains:

Each light chain interacts with one heavy chain to form a functional antigen-binding site. This pairing involves complementary interactions between the variable regions of both chains, ensuring precise and effective antigen recognition.

Analysis Methods for Antibody Heavy Chains and Light Chains

Antibody Sequencing

  • Mass Spectrometry (MS) Antibody Sequencing: Utilizes high-resolution mass spectrometry to sequence peptides derived from enzymatic digestion of antibody heavy and light chains. This method provides detailed information about the amino acid sequence, post-translational modifications, and structural variants.
  • Edman Degradation: A classical method for sequencing proteins, involving stepwise cleavage of amino acids from the N-terminus of the chain. Though less commonly used now due to automation and sensitivity limitations, it still provides reliable sequence data.

Protein Structure Analysis

  • X-ray Crystallography: Determines the three-dimensional structure of antibody heavy and light chains by analyzing diffraction patterns of X-rays passing through crystallized protein samples. Provides atomic-level details of antibody-antigen interactions and conformational changes.
  • Cryo-Electron Microscopy (Cryo-EM): Uses electron microscopy to visualize frozen hydrated specimens of antibody complexes. Allows for the determination of antibody structure at near-atomic resolution without the need for crystallization.

Functional Assays

  • Enzyme-Linked Immunosorbent Assay (ELISA): Quantifies and detects specific antibody heavy and light chains based on their interaction with antigens immobilized on a solid surface. Used for assessing antibody binding affinity, specificity, and concentration.
  • Surface Plasmon Resonance (SPR): Measures real-time interactions between antibody heavy and light chains and antigens immobilized on a sensor surface. Provides kinetic data on binding affinity, association, and dissociation rates.

Bioinformatics and Computational Modeling

  • Homology Modeling: Predicts the three-dimensional structure of antibody heavy and light chains based on known structures of related proteins. Useful for understanding structure-function relationships and designing engineered antibodies.
  • Sequence Alignment and Phylogenetic Analysis: Compares antibody heavy and light chain sequences across species or within individuals to identify conserved regions, mutations, and evolutionary relationships. Helps elucidate antibody diversity and evolution.

Reference

  1. Golpour, Monireh, et al. "The Perspective of Therapeutic Antibody Marketing in Iran: Trend and Estimation by 2025." Advances in pharmacological and pharmaceutical sciences 2021.1 (2021): 5569590.