Abstract
BackgroundMTMDAT is a program designed to facilitate analysis of mass spectrometry data of proteins and biomolecular complexes that are probed structurally by limited proteolysis. This approach can provide information about stable fragments of multidomain proteins, yield tertiary and quaternary structure data, and help determine the origin of stability changes at the amino acid residue level. Here, we introduce a pipeline between MTMDAT and HADDOCK, that facilitates protein-protein complex structure probing in a high-throughput and highly automated fashion.ResultsA new feature of MTMDAT allows for the direct identification of residues that are involved in complex formation by comparing the mass spectra of bound and unbound proteins after proteolysis. If 3D structures of the unbound components are available, this data can be used to define restraints for data-driven docking to calculate a model of the complex. We describe here a new implementation of MTMDAT, which includes a pipeline to the data-driven docking program HADDOCK, thus streamlining the entire procedure. This addition, together with usability improvements in MTMDAT, enables high-throughput modeling of protein complexes from mass spectrometry data. The algorithm has been validated by using the protein-protein interaction between the ubiquitin-binding domain of proteasome component Rpn13 and ubiquitin. The resulting structural model, based on restraints extracted by MTMDAT from limited proteolysis and modeled by HADDOCK, was compared to the published NMR structure, which relied on twelve unambiguous intermolecular NOE interactions. The MTMDAT-HADDOCK structure was of similar quality to structures generated using only chemical shift perturbation data derived by NMR titration experiments.ConclusionsThe new MTMDAT-HADDOCK pipeline enables direct high-throughput modeling of protein complexes from mass spectrometry data. MTMDAT-HADDOCK can be downloaded from http://www.ifm.liu.se/chemistry/molbiotech/maria_sunnerhagens_group/mtmdat/together with the manual and example files. The program is free for academic/non-commercial purposes.
Highlights
MTMDAT is a program designed to facilitate analysis of mass spectrometry data of proteins and biomolecular complexes that are probed structurally by limited proteolysis
To demonstrate that the quality of the protein complex structures obtained from limited proteolysis/mass spectrometry data can be competitive compared to structures generated with more classical restraints from chemical shift perturbations (CSP) acquired by nuclear magnetic resonance (NMR), we studied the complex of the proteasome subunit Rpn13 with ubiquitin
For deriving ambiguous interaction restraints (AIRs) from these files, a threshold of 20% was used for Rpn13, meaning that the relative cleavage propensity of a given residue in the complex must be at least 20% smaller than for Rpn13 in the absence of ubiquitin
Summary
MTMDAT is a program designed to facilitate analysis of mass spectrometry data of proteins and biomolecular complexes that are probed structurally by limited proteolysis. Chemical cross-linking in combination with mass spectrometry can reveal structural insight into proteins and their interactions [4,5,6,7,8], while the employment of radical probe mass spectrometry (RP-MS, [9]) evaluated by PROXIMO can yield structural models of protein complexes [10]. These methods are, very sophisticated, requiring expensive state-of-the-art equipment and expert knowledge. Time-consuming optimizations are often required, e.g. to find the right cross-linkers and conditions
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