CDMS Analysis of Intact 19S, 20S, 26S, and 30S Proteasomes: Evidence for Higher-Order 20S Assemblies at a Low pH

Abstract

Charge detection mass spectrometry (CDMS) was examined as a means of studying proteasomes. To this end, the following masses of the 20S, 19S, 26S, and 30S proteasomes from Saccharomyces cerevisiae (budding yeast) were measured: m(20S) = 738.8 ± 2.9 kDa, m(19S) = 926.2 ± 4.8 kDa, m(26S) = 1,637.0 ± 7.6 kDa, and m(30S) = 2,534.2 ± 10.8 kDa. Under some conditions, larger (20S) x (where x = 1 to ∼13) assemblies are observed; the 19S regulatory particle also oligomerizes, but to a lesser extent, forming (19S) x complexes (where x = 1 to 4, favoring the x = 3 trimer). The (20S) x oligomers are favored in vitro, as the pH of the solution is lowered (from 7.0 to 5.4, in a 20 mM ammonium acetate solution) and may be related to in vivo proteasome storage granules that are observed under carbon starvation. From measurements of m(20S) x (x = 1 to ∼13) species, it appears that each multimer retains all 28 proteins of the 20S complex subunit. Several types of structures that might explain the formation of (20S) x assemblies are considered. We stress that each structural type [hypothetical planar, raft-like geometries (where individual proteasomes associate through side-by-side interactions); elongated, rodlike geometries (where subunits are bound endto-end); and geometries that are roughly spherical (arising from aggregation through nonspecific subunit interactions)] is highly speculative but still interesting to consider, and a short discussion is provided. The utility of CDMS for characterizing proteasomes and related oligomers is discussed.

Adapting a trapped ion mobility spectrometry-Q-TOF for native mass spectrometry

“…30,33 Currently, nMS has been applied to measure macromolecules in the MDa mass range, such as adeno-associated virus capsids (3.5 -5 MDa), antibody-bound capsids species (5 -17 MDa), and oligomerized 20S proteasome (0.7 -9.6 MDa). [34][35][36][37] The Wysocki lab has demonstrated the application of SID, the only MS/MS that extensively dissociates MDa biomolecules, to investigate the structure of AAVs. 38 Here, we modified a commercial TIMS-Q-TOF mass spectrometer to study structures of high molecular weight (MW) protein complexes.…”

Adapting a trapped ion mobility spectrometry-Q-TOF mass spectrometer for native mass spectrometry

“…30,33 Currently, nMS has been applied to measure the structures of macromolecules in the MDa mass range, such as adeno-associated virus capsids (3.5 -5 MDa), antibody-bound capsids species (5 -17 MDa), and oligomerized 20S proteasome (0.7 -9.6 MDa), which are important for producing biotherapeutics. [34][35][36][37] The Wysocki lab has demonstrated that SID is currently the only activation method capable of extensively dissociating these MDa biomolecules, making it a valuable tool for investigating the structure of AAVs. 38 Here, we modified a commercial TIMS-Q-TOF mass spectrometer to study the structures of high molecular weight (MW) protein complexes.…”