The Use of Crosslinking Agents to Study Protein Interactions
Crosslinkers and Mass Spectrometry in Protein Structure and Protein Interaction Analysis

The Use of Crosslinking Agents to Study Protein Interactions

Covalently Bonding Interacting Proteins
When two or more proteins have specific affinity for one another that causes them to come together in biological systems, bioconjugation technology can provide the means for investigating those interactions. Most in vivo protein:protein binding is transient and occurs only briefly to facilitate signaling or metabolic function. Capturing or freezing these momentary contacts to study which proteins are involved and how they interact is a significant goal of proteomics research today.

Crosslinking reagents can provide the means for capturing protein:protein complexes by covalently bonding them together as they interact. The rapid reactivity of the common functional groups on crosslinkers allows even transient interactions to be frozen in place or weakly interacting molecules to be seized in a complex stable enough for isolation and characterization.

Targeting and Controlling Specificity of Crosslinking
The simple addition of homobifunctional or heterobifunctional crosslinkers to cell suspensions or cell lysates will cause many protein conjugates to be formed, not just those directly involved in the target protein:protein interaction. Many cell surface protein interactions have been studied using this “shotgun” approach, but the challenge in this technique is the analysis of data after complexes have been isolated.

To help solve these problems, more sophisticated crosslinker designs were created that incorporate photoreactive groups, which can be made to react at selected times and only in response to irradiation by UV light. Heterobifunctional crosslinkers with a thermoreactive group (spontaneously reactive) at one end and a photoreactive group on the other end can be reacted first through the thermoreactive end with a protein that can be used as bait for other interacting proteins. The modified protein is introduced into a sample and allowed to interact with other proteins. Then the sample is exposed to UV light, which causes the photoreactive end of the modified protein to covalently link to nearby molecules, thus “freezing” in place any interacting protein as a complex.

Photoreactive Crosslinkers
The use of photoreactive crosslinkers is preferred for studying protein interactions over methods that use standard bifunctional thermoreactive cross-linkers because photoreactive crosslinkers limit the formation of conjugate polymer artifacts. However, the downside of some photoreactive coupling methods is that the yield of conjugate formation is typically low. Particularly, many aryl azide groups undergo an inefficient ring-expansion reaction, which directs their reactivity exclusively toward amine groups, therefore limiting their utility for non-selective insertion into any neighboring protein structures. In addition, solvent reactions that quench the photoreactive intermediate often exceed reactions with a desired target.

Some photoreactive groups, such as halogen-substituted aryl azides and benzophenones, have much better conjugation yields and can efficiently capture interacting molecules. For instance, crosslinkers that incorporate a perfluoroazidobenzamido photoreactive group do not undergo ring expansion after photolyzing, thus they create a highly-reactive nitrene upon UV exposure that effectively couples to any protein structures nearby. An example of this type of photoreactive reagent is SFAD (Product # 27719 ), which has an amine-reactive sulfo-NHS-ester at one end and the halogen substituted phenylazide group at the other end.

Crosslinkers and Mass Spectrometry in Protein Structure and Protein Interaction Analysis
The ability to selectively conjugate two or more proteins together using cross-linking reagents permits the study of interacting proteins in complex mixtures. As the proteome becomes more defined, the interactions those protein molecules undergo will become increasingly important to understand. Pierce bioconjugation reagents are a critical factor in facilitating this knowledge. Additional information on these reagents and crosslinking chemistry in general is provided in the Protein Structure section.

Recently, in the drive to better understand protein structure and function, two powerful methods have been used in tandem to yield information for 3-D mapping of proteins and protein complexes.¹ The methods are Mass Spectrometry (MS) and protein cross-linking. Pierce Double-Agents cross-linking reagents have played an important role in the several studies published to date. Mass spectrometric analysis of the reaction products can yield low resolution three dimensional protein structure information giving insight into how a protein folds.² Analysis of interface sequences between interacting proteins yields insight into the composition and location of their respective molecular contact surfaces.

Popular Pierce cross-linking reagents with application in MS analysis:

• DST (Product #20589): Disuccinimidyl tartarate
• BS3 (Product # 21580): Bis(sulfosuccinimidyl) suberate
• Sulfo-EGS (Product # 21566): Ethylene glycol bis(sulfosuccinimidyl succinate
• DTSSP (Product #21578): 3,3'-Dithiobis(sulfosuccinimidyl propionate)

1. Sinz, A. (2003). Chemical cross-linking and mass spectrometry for mapping threedimemsional structures of proteins and protein complexes. J. Mass Spectrom. 38, 1225-1237.
2. Dihazi, G.H. and Sinz, A. (2003). Mapping low-resolution three-dimensional protein structures using chemical cross-linking and Fourier transform ion-cyclotron resonance mass spectrometry. Rapid Commun. Mass Spectrom. 17, 2005-2014.
3. Muller, D.R., et al. (2001). Isotope-tagged cross-linking reagents. A new tool in mass spectrometric protein interaction analysis. Anal. Chem. 73, 1927.
4. Pearson, K.M., et al. (2002). Intramolecular cross-linking experiments on cytochrome c and ribonuclease A using an isotope multiplet method. Rapid. Commun. Mass Spectrom. 16, 149.

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