Protein cross-linking reagents, fondly called “cross-linkers” by researchers, are molecules with two or more reactive ends that can attach themselves to specific functional groups (e.g., primary amines, carboxyls, sulfhydryls, carbohydrates, and carboxylic acids) on proteins and other biomolecules via a covalent bond.
Cross-linkers have an incredibly unique nature and can be used to stabilize the tertiary and quaternary structure of a particular protein or a protein-protein interaction, generate a conjugate that can be used in detection procedures, or facilitate the appropriate conditions for particular assays. These reagents play an important role in proteomic studies and can be used for numerous applications, which include:
- Structural and functional studies
- Protein and ligand-receptor interactions
- Antibody/immunotoxin production
- Cell membrane/cell surface studies
- Attaching or immobilizing proteins onto a solid support for protein analysis or affinity purification
- Preparation of protein-protein conjugates and secondary antibodies
- DNA/RNA to protein cross-linking
- Reactive group transfer, e.g., modifying target groups or adding space for ensuing coupling reactions
What Should You Consider When Choosing a Protein Cross-linker?
There are eight primary considerations when choosing an appropriate cross-linker to ensure the success of any bioconjugation experiment.
Reagent solubility
The solubility of a cross-linking reagent (as determined by its reactive group(s) and spacer arm composition) affects its ability to permeate into cells and conjugate hydrophobic proteins within membranes. Most cross-linkers are soluble in organic solvents (e.g., DMSO, DMF) but some applications necessitate the use of water-soluble cross-linkers.
Nature of reactive groups
The protein structure is incredibly complex, but surprisingly, there are only four functional groups that can be targeted for cross-linking techniques, namely primary amines, carboxyls, sulfhydryls, and carbonyls. One or more reactive groups exists for each of these target functional groups, so be careful in choosing which cross-linking reagent to use. Ideally, choose one that does not interfere with the functions of your target protein.
Aside from the nature of the functional group, also consider the number of functional groups on the surface of the protein. Ideally, use a lower cross-linker/protein ratio when there are several target groups and use a higher cross-linker/protein ratio if there are only a few potential targets. Keep the number of components at a minimum to facilitate a more accurate analysis.
Chemical specificity (homobifunctional or heterobifunctional)
Homobifunctional cross-linking reagents have identical reactive groups on both ends of the spacer arm. These are mostly used in single-step reactions and are exceptionally useful in creating intramolecular crosslinks, polymerizing molecules with like functional groups, and evaluating protein interactions. Some of the most popular homobifunctional cross-linking reagents include BMOE (bismaleimidoethane), DTME (dithiobismaleimidoethane), DSP (dithiobis succinimidyl propionate), DSs (disuccinimidyl suberate), and DST (disuccinimidyl tartrate).
Heterobifunctional cross-linking reagents have two distinct reactive groups on either end and are used in more controlled two-step reactions to reduce undesirable polymerization and cross-reaction. Some common examples include sulfo-SMCC, ANB-NOS (N-5-Azido-2-nitrobenzoyloxysuccinimide), ABH (p-Azidobenzoyl Hydrazide), and EMCH (N-(E-maleimidocaproyloxy)-succinimide ester).
Photoreactive or thermoreactive groups
Heterobifunctional cross-linking reagents can have a thermoreactive or spontaneously active group on one end and a photoreactive group (which is activated upon irradiation with UV light) on the other.
Photoreactive cross-linking reagents typically contain aryl azides (phenylazides) which form highly reactive aryl nitrenes upon exposure to light. These reagents can effectively capture interacting molecules and are extremely useful when there is insufficient information on the availability of thermoreactive functional groups. However, these reagents are not very efficient and may produce extremely low yields.
Note: Do not use acidic and reducing agents since they can inactivate aryl azide groups.
Length of the spacer arm
Spacer arm length refers to the distance between conjugated molecules, and may be classified as short (< 10 Å), medium (10.1–30 Å), or long (> 30 Å). Cross-linkers with short to medium spacer arms are suitable for intramolecular cross-linking, while longer spacer arms are ideal for intermolecular cross-linking. Using various spacer arm lengths is recommended to ensure a more comprehensive analysis of the protein structure.
Reversible or cleavable
A cleavable cross-linking reagent facilitates the easy release of cross-linked proteins from solid supports and lets the linkage be broken when needed (e.g., for further downstream applications), allowing the molecule to return to its original state. On the other hand, a reversible cross-linking reagent allows for the recovery and identification of interacting proteins.
Potential for further labeling
Labeling refers to any form of cross-linking which attaches a chemical group to a known protein (“bait”) to aid in the detection, identification, and purification of an unknown protein (“prey”). The label is then cleaved, leaving the label attached to the prey.
Reaction condition needed for conjugation
Cross-linking is usually performed under mild pH and buffer conditions to preserve the native structure of the protein complex. Optimal cross-linker/protein molar ratios for reactions, the degree of conjugation, and the number of functional groups on the protein’s surface must also be determined to obtain the desired results.
