Chemical Cross-Linking Mass Spectrometry for Studying Protein…

Chemical cross-linking is a mass spectrometric (CXMS) method for looking at protein complexes, which are an important part of all cellular processes — from signal transduction and metabolic pathways to the regulation of gene expression and transcription. Alterations in these interactions have led to such diseases as Huntington’s, Creutzfeldt-Jakob, and Alzheimer’s. There are a multitude of different approaches that have been used to detect and characterize these complexes, with the most common being X-ray crystallography and NMR. However, these have their drawbacks because they are limited by the quantity and purity required for such analyses and the size and homogeneity of the complexes formed.¹

CXMS involves forming covalent bonds between two interacting proteins so that the interaction between two amino acids is preserved. The distance between the two amino acids varies depending on the length of linker used as well as the specificity of the amino acids involved. Libraries have been created by Thermo Scientific and other large companies for this particular use.²The covalent linkages can be homofunctional, such as Lys/Lys, or bifunctional, such as Lys/Glu. The spatial distances between the active sites can range from the zero length carbodiimide to longer and longer linkers. The range of linker length allows for spatial distances to be calculated, working as a set of molecular rulers and allowing for distances between amino acids that are relevant to the tertiary and quaternary structures of proteins and their complexes to be identified.³

At the forefront of CXMS is Dr. David R. Goodlett and his lab, which have been working on this since at least 2006. His initial work involved cytochrome P450 2E1 and cytochrome b5 as tests for methodology and validation of analysis. Using this simple complex, the Goodlett group were able to identify, assign, and validate six locations between the two proteins where they were in proximity to be covalently linked together using a zero-length linker.^1,2,4 Recently, this approach was used by Goodlett et al. to identify and validate the subunits that make up the proteasome core particle in Haloferax volcanii. The results showed two of four possible combinations of the subunits in the proteasome core. The approach was simple, involving cross-linking the proteasome, dialysis to remove salts, and digestion with trypsin, which was subsequently followed by LC-MS/MS on a hybrid linear ion trap (Thermo Scientific) with a nanoAcquity HPLC (Waters). Peptides were eluted off the precolumn and column, rejecting any peptides with a charge of 1, 2, or 3, allowing the large cross-linked peptides to have priority.³ The rejection of smaller charge state peptides removed the need for a purification step prior to MS analysis.³

One drawback to this type of work is the difficulty in acquiring software able to decode, interpret, and validate the data. In many instances, groups have resorted to creating their own software; however, some currently available software can be used in this manner with some modifications. Software such as Phenyx (GeneBio) can be used to identify the proteins but only after creating a database of potential cross-linked peptides, which can be done using XComb (GeneBio).⁴ Integration of personal databases is also possible with in-house Mascot (Matrix Science), which would allow for the identification of modified peptides.² These databases and searches require previous information about the interaction to be known so that the databases can be created. Any other interactions with proteins that were not inserted into the database will not be accounted for. There is another useful online database STRING that provides a network of protein-protein interactions.^5,6 It is hoped that in the future some of the most common search algorithms will allow for this type of modification without having to create new databases for the sole purpose of finding these modifications.

References

1. Singh, P., et al. (2008) ‘Characterization of protein cross-links via mass spectrometry and an open-modification search strategy‘, Analytical Chemistry, 80 (22), (pp. 8799–8806)

2. McIlwain, S., et al. (2010) ‘Detecting cross-linked peptides by searching against a database of cross-linked peptide pairs‘, Journal of Proteome Research, 9 (5), (pp. 2488–2495)

3. Karadzic, I., et al. (2012) ‘Chemical cross-linking, mass spectrometry, and in silico modeling of proteasomal 20S core particles of the haloarchaeon Haloferax volcanii‘, Proteomics, 12 (11), (pp. 1806–1814)

4. Gao, Q., et al. (2006) ‘Pro-CrossLink. Software tool for protein cross-linking and masss spectrometry‘, Analytical Chemistry, 78 (7), (pp. 2145–2149)

5. http://string-db.org/

6. Franceschini, A., et al. (2013) ‘STRING v9.1: protein-protein interaction networks, with increased coverage and integration’, Nucleic Acids Res, 41 (D1), (pp. D808-D815)