TOP-DOWN SEQUENCING OF CROSSLINKED PROTEINS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. During the past few years mass spectrometry has emerged as leading technology for proteomics applications thanks to the continuous development of new methodologies and better instrumentation. Among these, a branch known as Structural Proteomics has grown into a powerful technique, capable of offering a wide variety of approaches to low resolution characterization of protein structure by combining protein chemistry with modern mass spectrometry. This approach has proven capable of delivering information that can be complementary to those obtained by high resolution techniques. Chemical crosslinking [1] is one of the most common methodologies employed in the analysis of protein complexes. In this work we explore limits and possibilities of a top-down [2,3] approach to the study of protein interactions and quaternary structure. Lyophilized human hemoglobin or recombinant transthyretin was dissolved in sodium acetate buffer (pH 7.5). Aliquots of Bis[sulfosuccinimidyl]suberate (BS3) (5 mM, 10 mM, 25 mM, 50 mM, 100 mM) were added to different reaction mixtures, and the reactions were quenched with NH4HCO3 after 5, 15, 30, 60, 120 min. Samples were analyzed as is with a Thermo-Fisher LTQ-Orbitrap "Discovery" mass spectrometer using an Advion Triversa NanoMate ESI source or the Bruker SolariX 12-T FTMS. Tandem mass spectra were generated by LTQ-CID and HCD in the C-trap or nozzle-skimmer dissociation (NSD) on the LTQ-Orbitrap or by SORI-CID , ECT or ETD on the SolariX and were deconvoluted using the proprietary software for each instrument. Fragment mass lists were analyzed using BUPID-Topdown (Boston University Protein Identifier-Topdown), a custom-programmed software algorithm written in-house. In order to assess the reaction kinetics and follow the formation different species, different reaction mixtures, with increasing molar excess of crosslinking agent, were followed by a time course analysis. With increasing reaction time, the samples showed a growing degree of complexity, and thus indicated the occurrence of a variety of combinations of protein modification and both intra- and intermolecular crosslinking events, ultimately leading to the disappearance of the sample signal. Among the analyzed mixtures, we were able to observe the formation of only homo-dimers of the alpha- and beta-subunits of hemoglobin, no hetero-dimer was ever detected. We isolated different charge states of the crosslinked homodimers and with the combination of different fragmentation techniques, applied to different charge states, we were able to locate the identify the specific amino acids involved in the crosslinking reaction, and thus the region of the protein involved in the interactions. Our results are consistent with X-ray structural determinations of hemoglobin and TTR. They also show that the well established technique of protein crosslinking for conformational analysis can be performed by a top-down methodology. With this approach we have been able to locate the regions of protein interaction by direct analysis of protein, and to analyze the reaction behavior in order to choose the optimal reaction conditions, in order to preserve the native tertiary structure of the protein under analysis. This methodology no only offers the possibility for quaternary structural analysis of proteins even when crystals are not available, but also expedites the normal procedures employed for chemical crosslinking, including gel based analysis that requires larger amounts of sample and offer a lesser degree of control over the reaction conditions. We have now extended the study to include the tetrameric protein transthyretin, and have again succeeded in finding crosslinking sites. To obtain the best results, these experiments are now being carried out on the newly installed Bruker 12-T FTMS. In addition, chromatographic methods for enriching the crosslinked products have been developed. 1.Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions. A. Sinz, Mass Spectrom Rev. 2006 25(4):663-82 2.Top down' versus 'bottom up' protein characterization by tandem high-resolution mass spectrometry. Kelleher NL, Lin HY, Valsakovic GA, Aaserud DJ, Fridrikson EK, Beavil A, Holowka D, Gould HJ, Baird B, McLafferty FW. J Am Chem Soc 1999, 121:806812 3.A top down approach to protein structural studies using chemical cross-linking and Fourier transform mass spectrometry. Kruppa GH, Schoeninger JS, Young MM. Rapid Commun Mass Spectrom 2003, 17:155 162