Crosslinking is the process of chemically joining two or more molecules by a covalent bond. The technique, often called bioconjugation when referring to its use with proteins and other biomolecules, is an essential component of many proteomics methods, including creation of detectable probes for Western blotting and ELISA and strategies for investigating protein structure and interactions.

Crosslinking reagents (or crosslinkers) are molecules that contain two or more reactive ends capable or chemically attaching to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other molecules. This article describes the chemistry and variety of crosslinkers that exist.



Overview of Crosslinker Structure and Chemistry

Protein Functional Groups

Despite the complexity of protein structure, including composition and sequence of 20 different amino acids, only a small number of protein functional groups comprise selectable targets for practical bioconjugation methods. In fact, just four protein chemical targets account for the vast majority of crosslinking and chemical modification techniques:

  • Primary amines (–NH2): This group exists at the N-terminus of each polypeptide chain (called the alpha-amine) and in the side chain of lysine (Lys, K) residues (called the epsilon-amine). Because of its positive charge at physiologic conditions, primary amines are usually outward-facing (i.e, on the outer surface) of proteins; thus, they are usually accessible for conjugation without denaturing protein structure.
  • Carboxyls (–COOH): This group exists at the C-terminus of each polypeptide chain and in the side chains of aspartic acid (Asp, D) and glutamic acid (Glu, E). Like primary amines, carboxyls are usually on the surface of protein structure.
  • Sulfhydryls (–SH): This group exists in the side chain of cysteine (Cys, C). Often, as part of a protein's secondary or tertiary structure, cysteines are joined together between their side chains via disulfide bonds (–S–S–). These must be reduced to sulfhydryls to make them available for crosslinking by most types of reactive groups.
  • Carbonyls (–CHO): Ketone or aldehyde groups can be created in glycoproteins by oxidizing the polysaccharide post-translational modifications (glycosylation) with sodium meta-periodate.

Crosslinking Reagents Technical Handbook

This 45-page guide is of value to the novice as well as those who have previous experience with crosslinking reagents. It begins with a basic discussion on crosslinking and the reagents that are used. The guide also contains a discussion on various applications where crosslinking has been applied, including the powerful label-transfer technique for identifying or confirming protein interactions. Crosslinking chemistry is addressed in an easy-to-follow format designed to convey the important information you need without getting lost in details. Each Pierce crosslinking reagent is shown along with it's structure, molecular weight, spacer arm length and chemical reactivity. The handbook concludes with a list of excellent references on cross-linker use and a glossary of common crosslinking terms.

Crosslinker Reactive Groups

A number of chemical reactive groups have been characterized and used to target the main kinds of protein functional groups. Many different crosslinking reagents can be synthesized when different combinations of two are more of these reactive groups are incorporated into one molecule. When combined with different sizes and types of chemical "backbones" (called spacer arms because they define the distance between respective reactive ends), the number of possible crosslinking compounds is enormous.

Popular crosslinker reactive groups for protein conjugation Reactivity class titles link to specific sections of this article. Italicized chemical group names are not specifically discussed in this article.
Reactivity Class Chemical Group
Carboxyl-to-amine reactive groups Carbodiimide (e.g., EDC)
Amine-reactive groups NHS ester
Imidoester
Pentafluorophenyl ester
Hydroxymethyl phosphine
Sulfhydryl-reactive groups Maleimide
Haloacetyl (Bromo- or Iodo-)
Pyridyldisulfide
Thiosulfonate
Vinylsulfone
Aldehyde-reactive groups
i.e., oxidized sugars (carbonyls)
Hydrazide
Alkoxyamine
Photoreactive groups
i.e., nonselective, random insertion
Diazirine
Aryl Azide
Hydroxyl (nonaqueous)-reactive groups Isocyanate

Bioconjugate Techniques, 3rd Edition

Bioconjugate Techniques, 3rd Edition (2013) by Greg T. Hermanson is a major update to a book that is widely recognized as the definitive reference guide to the field of bioconjugation.

Bioconjugate Techniques is a complete textbook and protocols-manual for life scientists wishing to learn and master the biomolecular crosslinking, labeling and immobilization techniques that form the basis of many laboratory applications. The book is also an exhaustive and robust reference for researchers looking to develop novel conjugation strategies for entirely new applications. It also contains an extensive introduction to the field of bioconjugation that covers all of the major applications of the technology used in diverse scientific disciplines as well as containing tips for designing the optimal bioconjugate for any purpose.


Selecting Crosslinkers

Crosslinkers are selected on the basis of their chemical reactivities (i.e., specificity for particular function groups) and other chemical properties that affect their behavior in different applications:

  • Chemical specificity refers to the reactive target(s) of the crosslinker's reactive ends. A general consideration is whether the reagent has the same or different reactive groups at either end (termed homobifunctional and heterobifunctional, respectively; see below).
  • Spacer arm length refers to the molecular span of a crosslinker (i.e., the distance between conjugated molecules). A related consideration is whether the arm is cleavable (i.e., whether the linkage can be reversed or broken when desired).
  • Water-solubility and cell membrane permeability of a crosslinker affect whether it can permeate into cells and/or crosslink hydrophobic proteins within membranes. These properties are determined by the composition of the spacer arm and/or reactive group.
  • Spontaneously reactive or photoreactive groups in a crosslinker affect whether it react as soon as it is added to a sample or can be activated at a specific time by exposure to UV light.

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Homobifunctional and Heterobifunctional Crosslinkers

Crosslinkers can be classified as homobifunctional or heterobifunctional.

Homobifunctional crosslinkers have identical reactive groups at either end of a spacer arm, and generally they must be used in one-step reaction procedures to randomly "fix" or polymerize molecules containing like functional groups. For example, adding an amine-to-amine crosslinker to a cell lysate will result in random conjugation of protein subunits, interacting proteins and any other polypeptides whose lysine side chains happen to be near each other in the solution. This is ideal for capturing a "snapshot" of all protein interactions but cannot provide the precision needed for other types of crosslinking applications. For example, when preparing an antibody-enzyme conjugate, the goal is to link one to several enzyme molecules to each molecule of antibody without causing any antibody-to-antibody linkages to form. This is not possible with homobifunctional crosslinkers.

Homobifunctional crosslinker example. DSS is a popular, simple crosslinker that has identical amine-reactive NHS-ester groups at either end of a short spacer arm. The spacer arm length (11.4 angstroms) is the final maximum molecular distance between conjugated molecules (i.e., nitrogens of the target amines).

21655-DSS-stc

Heterobifunctional crosslinkers possess different reactive groups at either end. These reagents not only allow for single-step conjugation of molecules that have the respective target functional groups, but they also allow for sequential (two-step) conjugations that minimize undesirable polymerization or self-conjugation. In sequential procedures, heterobifunctional reagents are reacted with one protein using the most labile group of the crosslinker first. After removing excess nonreacted crosslinker, the modified first protein is added to a solution containing the second protein where reaction through the second reactive group of the crosslinker occurs. The most widely-used heterobifunctional crosslinkers are those having an amine-reactive succinimidyl ester (i.e., NHS ester) at one end and a sulfhydryl-reactive group (e.g., maleimide) on the other end. Because the NHS-ester group is less stable in aqueous solution, it is usually reacted to one protein first. If the second protein does not have available native sulfhydryl groups, they can be added in a separate prior step using sulfhydryl-addition reagents.

22322-Sulfo-SMCC-stc
Heterobifunctional crosslinker example. Sulfo-SMCC is a popular crosslinker that has an amine-reactive sulfo-NHS-ester group (left) at one end and a sulfhydryl reactive maleimide group (right) at the opposite end of a cyclohexane spacer arm. This allows for sequential, two-step conjugation procedures.

General Reaction Conditions

In many applications, it is necessary to maintain the native structure of the protein complex, so crosslinking is most often performed using near-physiologic conditions. Optimal crosslinker-to-protein molar ratios for reactions must be determined empirically, although product instructions for individual reagents generally contain guidelines and recommendations for common applications.

Depending on the application, the degree of conjugation is an important factor. For example, when preparing immunogen conjugates, a high degree of conjugation is desired to increase the immunogenicity of the antigen. However, when conjugating to an antibody or an enzyme, a low-to-moderate degree of conjugation may be optimal so that biological activity of the protein is retained.

The number of functional groups on the protein’s surface is also important to consider. If there are numerous target groups, a lower crosslinker-to-protein ratio can be used. For a limited number of potential targets, a higher crosslinker- to-protein ratio may be required. Furthermore, the number of components should be kept low or to a minimum because conjugates consisting of more than two components are difficult to analyze and provide less information on spatial arrangements of protein subunits.


Reactive Group Chemistries
Carboxyl-to-Amine Crosslinker Groups

Carbodiimides (EDC and DCC)

EDC and other carbodiimides are zero-length crosslinkers; they cause direct conjugation of carboxylates (–COOH) to primary amines (–NH2) without becoming part of the final crosslink (amide bond) between target molecules.

01-Carbodiimide-stc

EDC crosslinking reactions must be performed in conditions devoid of extraneous carboxyls and amines. Acidic (pH 4.5 to 5.5) MES buffer (4-morpholino-ethane-sulfonic acid) is most effective, but phosphate buffers at pH ≤ 7.2 are also compatible with the reaction chemistry. N-hydroxysuccinimide (NHS) or its water-soluble analog (Sulfo-NHS) is often included in EDC coupling protocols to improve efficiency or to create a more stable, amine-reactive intermediate (see next section).

Because peptides and proteins contain multiple carboxyls and amines, direct EDC-mediated crosslinking usually causes random polymerization of polypeptides. Nevertheless, this reaction chemistry is used widely in immobilization procedures (e.g., attaching proteins to a carboxylated surface) and in immunogen preparation (e.g., attaching a small peptide to a large carrier protein).

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Amine-Reactive Crosslinker Groups

N-Hydroxysuccinimide Esters (NHS Esters)

NHS esters are reactive groups formed by EDC-activation of carboxylate molecules (see previous section). NHS ester-activated crosslinkers and labeling compounds react with primary amines in slightly alkaline conditions (pH 7.2-8.5) to yield stable amide bonds. The reaction releases N-hydroxysuccinimide (MW 115), which can be removed easily by dialysis or desalting.

02-NHS-Ester-stc

NHS-ester crosslinking reactions are usually performed in phosphate buffer at pH 7.2-8.0 for 0.5 to 4 hours at room temperature or 4°C. Primary amine buffers such as Tris (TBS) are not compatible because they compete for reaction; however, in some procedures, it is useful to add Tris or glycine buffer at the end of a conjugation procedure to quench (stop) the reaction.

Sulfo-NHS esters are identical to NHS esters except that they contain a sulfonate (–SO3) group on the N-hydroxysuccinimide ring. This charged group has no effect on the reaction chemistry, but it does tend to increased the water-solubilty of crosslinkers containing them. In addition, the charged group prevents Sulfo-NHS crosslinkers from permeating cell membranes, enabling them to be used for cell surface crosslinking methods.


Imidoesters

Imidoester crosslinkers react with primary amines to form amidine bonds. To ensure specificity for primary amines, imidoester reactions are best done in amine-free, alkaline conditions (pH 10), such as with borate buffer.

03-Imidoester-stc

Because the resulting amidine bond is protonated, the crosslink has a positive charge at physiological pH, much like the primary amine which it replaced. For this reason, imidoester crosslinkers have been used to study protein structure and molecular associations in membranes and to immobilize proteins onto solid-phase supports while preserving the isoelectric point (pI) of the native protein. Although imidoesters are still used in certain procedures, the amidine bonds formed are reversible at high pH. Therefore, the more stable and efficient NHS-ester crosslinkers have steadily replaced them in most applications.


Sulfhydryl-Reactive Crosslinker Groups
Maleimides

Maleimide-activated crosslinkers and labeling reagents react specifically with sulfhydryl groups (–SH) at near neutral conditions (pH 6.5-7.5) to form stable thioether linkages. Disulfide bonds in protein structures (e.g., between cysteines) must be reduced to free thiols (sulfhydryls) to react with maleimide reagents. Extraneous thiols (most reducing agents) must be excluded from maleimide reaction buffers, because they will compete for coupling sites.

04-Maleimide-stc

Short homobifunctional maleimide crosslinkers enable disulfide bridges in protein structures to be converted to permanent, irreducible linkages between cysteines. More commonly, the maleimide chemistry is used in combination with amine-reactive NHS-ester chemistry in the form of heterobifunctional crosslinkers that enable controlled, two-step conjugation of purified peptides and/or proteins.


Haloacetyls

Most haloacetyl crosslinkers contain an iodoacetyl or a bromoacetyl group. Haloacetyls react with sulfhydryl groups at physiologic to alkaline conditions (pH 7.2 to 9), resulting in stable thioether linkages. To limit free iodine generation, which has the potential to react with tyrosine, histidine and tryptophan residues, perform iodoacetyl reactions in the dark.

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Pyridyl Disulfides

Pyridyl disulfides react with sulfhydryl groups over a broad pH range to form disulfide bonds. As such, conjugates prepared using these crosslinkers are cleavable with typical disulfide reducing agents, such as dithiothreitol (DTT).

06-Pyridyldithiol-stc

During the reaction, a disulfide exchange occurs between the –SH group of the target molecule and the 2-pyridyldithiol group of the crosslinker. Pyridine-2-thione (MW 111; λmax 343nm) is released as a byproduct that can be monitored spectrophotometrically and removed from protein conjugates by dialysis or desalting.


Carbonyl-Reactive Crosslinker Groups
Hydrazides

Carbonyls (aldehydes and ketones) can be produced in glycoproteins and other polysaccharide-containing molecules by mild oxidation of certain sugar glycols using sodium meta-periodate. Hydrazide-activated crosslinkers and labeling compounds will then conjugate with these carbonyls at pH 5 to 7, resulting in formation of hydrazone bonds.

07-Hydrazide-stc

Hydrazide chemistry is useful for labeling, immobilizing or conjugating glycoproteins through glycosylation sites, which are often (as with most polyclonal antibodies) located at domains away from the key binding sites whose function one wishes to preserve.


Alkoxyamines

Although not currently as popular or common as hydrazide reagents, alkoxyamine compounds conjugate to carbonyls (aldehydes and ketones) in much the same manner as hydrazides.

07-Alkoxyamine-stc

Photoreactive Crosslinker Groups
Aryl Azides

Photoreactive reagents are chemically inert compounds that become reactive when exposed to ultraviolet or visible light. Historically, aryl azides (also called phenylazides) have been the most popular photoreactive chemical group used in crosslinking and labeling reagents.

08-Aryl-Azide-stc

When an aryl azide compound is exposed to UV light, it forms a nitrene group that can initiate addition reactions with double bonds or insertion into C-H and N-H sites or can undergo ring expansion to react with a nucleophile (e.g., primary amine). Reactions can be performed in a variety of amine-free buffer conditions to conjugate proteins or even molecules devoid of the usual functional group "handles".

Photoreactive reagents are most often used as heterobifunctional crosslinkers to capture binding partner interactions. A purified bait protein is labeled with the crosslinker using the amine- or sulfhydryl-reactive end. Then this labeled protein is added to a lysate sample and allowed to bind its interactor. Finally, photo-activation with UV light initiates conjugation via the phenyl azide group.


Diazirines

Diazirines are a newer class of photo-activatable chemical groups that are being incorporated into crosslinking and labeling reagents. The diazirine (azipentanoate) moiety has better photostability than phenyl azide groups, and it is more easily and efficiently activated with long-wave UV light (330-370 nm).

09-Diazirine-stc

Photo-activation of diazirine creates reactive carbene intermediates. Such intermediates can form covalent bonds through addition reactions with any amino acid side chain or peptide backbone at distances corresponding to the spacer arm lengths of the particular reagent. Diazirine-analogs of amino acids can be incorporated into protein structures by translation, enabling specific recombinant proteins to be activated as the crosslinker.


Chemoselective Ligation Groups
Staudinger Reagent Pairs

Chemoselective ligation refers to the use of mutually specific pairs of conjugation reagents. Staudinger ligation reagents are pairs of metabolic or chemical labeling compounds that have azide and phosphine groups, respectively. In other words, these reactive groups recognize each other but not any natural or endogenous biomolecules in typical samples. Thus, when respective pairs of target biomolecules are labeled with these two groups, they will conjugate to one another with high specificity when combined. This specialized form of crosslinking is most often used for in vivo metabolic labeling.

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