Bortezomib Inhibits Open Configurations of the 20S Proteasome.

Bortezomib, a small dipeptide-like molecule, is a proteasome inhibitor used widely in the treatment of myeloma and lymphoma. This molecule reacts with threonine side chains near the center of the 20S proteasome and disrupts proteostasis by blocking enzymatic sites that are responsible for protein degradation. In this work, we use novel mass-spectrometry-based techniques to examine the influence of bortezomib on the structures and stabilities of the 20S core particle.

Resolving Hidden Solution Conformations of Hemoglobin Using IMS-IMS on a Cyclic Instrument.

Ion mobility spectrometry-mass spectrometry (IMS-MS) experiments on a cyclic IMS instrument were used to examine heterogeneous distributions of structures found in the 15+ to 18+ charge states of the hemoglobin tetramer (Hb). The resolving power of IMS measurements is known to increase with increasing drift-region length. This effect is not significant for Hb charge states as peaks were shown to broaden with increasing drift-region length.

Stability of 20S Proteasome Configurations: Preopening the Axial Gate.

Mass spectrometry studies of the stability of the 20S proteasome from 11 to 55 °C reveal a series of related configurations and coupled transitions that appear to be associated with opening of the proteolytic core. We find no evidence for dissociation, and all transitions are reversible. A thermodynamic analysis indicates that configurations fall into three general types of structures: enthalpically stabilized, tightly closed (observed as the +54 to +58 charge states) configurations; high-entropy (+60 to +66) states that are proposed as precursors to pore opening; and larger (+70 to +79) partially and fully open pore structures.

Thermal Analysis of a Mixture of Ribosomal Proteins by vT-ESI-MS: Toward a Parallel Approach for Characterizing the .

The thermal stabilities of endogenous, intact proteins and protein assemblies in complex mixtures were characterized in parallel by means of variable-temperature electrospray ionization coupled to mass spectrometry (vT-ESI-MS). The method is demonstrated by directly measuring the melting transitions of seven proteins from a mixture of proteins derived from ribosomes. A proof-of-concept measurement of a fraction of an lysate is provided to extend this approach to characterize the thermal stability of a proteome.

Understanding the Thermal Denaturation of Myoglobin with IMS-MS: Evidence for Multiple Stable Structures and Trapped Pre-equilibrium States.

Thermal denaturation of holomyoglobin (hMb) in solution (10 mM ammonium acetate at pH = 4.5, 6.8, and 9.