Protein interactions reagents

Figure 18.1 A trifunctional reagent for studying protein interactions by mass spec. The bis-NHS ester arms crosslink interacting proteins, while the discrete PEG-containing biotin arm can be used to isolate or detect the conjugates using (strept)avidin reagents.

Figure 28.3 The homobifunctional crosslinkers BS2G and BS3 can be used to capture protein interactions through amide bond formation. The deuterium-labeled analogs of these reagents can be used to differentiate...

The use of PIR compounds to study protein interactions is a significant advance over the use of standard homobifunctional crosslinkers. The unique design of the PIR reagent facilitates deconvolution of putative protein interaction complexes through a simplified mass spec analysis. The software can ignore all irrelevant peak data and just focus analysis on the two labeled peptide peaks, which accompany the reporter signal of appropriate mass. This greatly simplifies the bioinformatics of data analysis and provides definitive conformation of protein-protein crosslinks. 

The following protocol is designed for treating cells with the PIR reagent to study protein interactions in vivo. It is based on the method of Tang et al. (2005). The use of the PIR compound to treat intact cells results in the crosslinking of proteins both on the cell surface and within the cell, which indicates that the reagent is able to cross the cell membrane. 

Figure 28.13 A sulfo-SBED-captured protein interaction can be released using DTT to cleave the disulfide within the cross-bridge leading to the bait protein. The result transfers the biotin label to the unknown interacting protein. The biotin tag thus allows the interacting protein to be detected or isolated using (strept)avidin reagents.

Alley, S.C., Ishmael, F.T., Jones, A.D., and Benkovic, S.J. (2000) Mapping protein-protein interactions in the bacteriophage T4 DNA polymerase holoenzyme using a novel trifunctional photo-cross-linking and affinity reagent./. Am. Chem. Soc. 122, 6126-6127. 

Mullet D.R. et al. (2001) Isotope-tagged cross-linking reagents. A new tool in mass spectrometric protein interaction analysis. Anal. Chem. 73, 1927-1934. 

Traviglia, S.L., Datwyler, S.A., and Meares, C.F. (1999) Mapping protein-protein interactions with a library of tethered cutting reagents The binding site of sigma (70) on Escherichia coli RNA polymerase. Biochemistry 38, 4259-4265. 

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