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Protein Interaction Networks: Reading Between the Lines

SBKB [doi:10.1038/sbkb.2012.136]
Technical Highlight - April 2013
Short description: An additional separation is added to LC-MS to obtain more information about quaternary structure of complexes.

LC-IMS-MS analysis results in signals separated by m/z (y axis) and drift time (x axis). Doublets are signals from heavy/light peptide pairs, but the triplet (shown above) is from a cross-linked homodimeric peptide (signals are from light-light, light-heavy, heavy-light and heavy-heavy pairs, giving a 1:2:1 ratio). Figure courtesy of Joshua Adkins.

Structural proteomics efforts have greatly benefited from the combination of cross-linking and mass spectrometry (MS). In the presence of a cross-linker, adjacent portions of proteins are linked covalently, and subsequent MS analysis identifies those neighboring fragments, providing clues about overall structure.

Complexes with multiple copies of the same protein, however, present the problem of distinguishing intra- from intermolecular cross-links: unless the identified peptides have overlapping, non-repeated sequences, the distinction cannot be made. This challenge has been addressed by mixing heavy-isotope labeled and unlabeled proteins, such that differentially labeled peptides, when cross-linked, result in a distinct MS signature. This approach, mixed-isotope cross-linking (MIX), has now been improved to uniquely identify more intermolecular cross-links.

Adkins and colleagues (Program for the Characterization of Secreted Effector Proteins partnership with MCSG) added ion mobility spectrometry (IMS) to the traditional liquid chromatography (LC)-MS analysis. A mixture of heavy isotope-labeled and unlabeled proteins was allowed to equilibrate in complexes, which was then cross-linked and digested. The resulting peptides were first separated by LC, then by IMS based on ion drift time, and finally by MS based on mass-to-charge ratio (m/z). In contrast to doublet signals arising from the heavy and light forms of unlinked peptides, cross-linked peptides have a quadruplet signal in a 1:1:1:1 ratio; homodimeric peptide pairs have a triplet signal in a 1:2:1 ratio.

LC-IMS-MS data are analogous to a two-dimensional gel, similarly allowing more information to be gleaned: the additional separation reveals data obscured in the MS-only spectrum. Indeed, comparing LC-IMS-MS data and simulated LC-MS data, the authors found that in the LC-MS-only data, signals from cross-linked peptides overlapped with those for unrelated, interfering peaks.

In this proof-of-principle work with homodimeric proteins, many multiplet peaks are distinguishable by sight. The group also developed a software application, called Cross-linking IMS, to automate the search and data analyses, a feature that will be critical for future high-throughput work with large protein complexes. To analyze such complexes using MIX-LC-IMS-MS, the authors suggest MIX-labeling a subset of subunits, or isotopically labeling the cross-linker. With any of these permutations, the additional IMS separation allows confident identification of more peptides.

Irene Kaganman


  1. E.D. Merkley et al. Mixed-Isotope Labeling with LC-IMS-MS for Characterization of Protein–Protein Interactions by Chemical Cross-Linking.
    J. Am. Soc. Mass Spectrom. (19 February 2013). doi:10.1007/s13361-012-0565-x

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