Direct Quantification of Gadolinium Retention in Young Patients by ICP-MS Analysis of Extracted Teeth.

In this 10-patient prospective pilot study, we show feasibility of pragmatic, direct ex vivo measurement of gadolinium retention from group II gadolinium-based contrast agents (GBCAs) in young patients following routine tooth extraction. This noninvasive method may support future research attempting to understand the link between GBCA exposure and clinical outcomes.

BoGH13ASus from Bacteroides ovatus represents a novel α-amylase used for Bacteroides starch breakdown in the human gut.

Members of the Bacteroidetes phylum in the human colon deploy an extensive number of proteins to capture and degrade polysaccharides. Operons devoted to glycan breakdown and uptake are termed polysaccharide utilization loci or PUL. The starch utilization system (Sus) is one such PUL and was initially described in Bacteroides thetaiotaomicron (Bt). BtSus is highly conserved across many species, except for its extracellular α-amylase, SusG. In this work, we show that the Bacteroides ovatus (Bo) extracellular α-amylase, BoGH13ASus, is distinguished from SusG in its evolutionary origin and its domain architecture and by being the most prevalent form in Bacteroidetes Sus. BoGH13ASus is the founding member of both a novel subfamily in the glycoside hydrolase family 13, GH13_47, and a novel carbohydrate-binding module, CBM98. The BoGH13ASus CBM98-CBM48-GH13_47 architecture differs from the CBM58 embedded within the GH13_36 of SusG. These domains adopt a distinct spatial orientation and invoke a different association with the outer membrane. The BoCBM98 binding site is required for Bo growth on polysaccharides and optimal enzymatic degradation thereof. Finally, the BoGH13ASus structure features bound Ca2+ and Mn2+ ions, the latter of which is novel for an α-amylase. Little is known about the impact of Mn2+ on gut bacterial function, much less on polysaccharide consumption, but Mn2+ addition to Bt expressing BoGH13ASus specifically enhances growth on starch. Further understanding of bacterial starch degradation signatures will enable more tailored prebiotic and pharmaceutical approaches that increase starch flux to the gut.

Ion Mobility-Mass Spectrometry Reveals Highly-Compact Intermediates in the Collision Induced Dissociation of Charge-Reduced Protein Complexes.

Protocols that aim to construct complete models of multiprotein complexes based on ion mobility and mass spectrometry data are becoming an important element of integrative structural biology efforts. However, the usefulness of such data is predicated, in part, on an ability to measure individual subunits removed from the complex while maintaining a compact/folded state. Gas-phase dissociation of intact complexes using collision induced dissociation is a potentially promising pathway for acquiring such protein monomer size information, but most product ions produced are possessed of high charge states and elongated/string-like conformations that are not useful in protein complex modeling. It has previously been demonstrated that the collision induced dissociation of charge-reduced protein complexes can produce compact subunit product ions; however, their formation mechanism is not well understood. Here, we present new experimental evidence for the avidin (64 kDa) and aldolase (157 kDa) tetramers that demonstrates significant complex remodeling during the dissociation of charge-reduced assemblies. Detailed analysis and modeling indicates that highly compact intermediates are accessed during the dissociation process by both complexes. Here, we present putative pathways that describe the formation of such ions, as well as discuss the broader significance of such data for structural biology applications moving forward.

Ion mobility-mass spectrometry of charge-reduced protein complexes reveals general trends in the collisional ejection of compact subunits.

Multiprotein complexes have been shown to play critical roles across a wide range of cellular functions, but most probes of protein quaternary structure are limited in their ability to analyze complex mixtures and polydisperse structures using small amounts of total protein. Ion mobility-mass spectrometry offers a solution to many of these challenges, but relies upon gas-phase measurements of intact multiprotein complexes, subcomplexes, and subunits that correlate well with solution structures. The greatest bottleneck in such workflows is the generation of representative subcomplexes and subunits. Collisional activation of complexes can act to produce product ions reflective of protein complex composition, but such product ions are typically challenging to interpret in terms of their relationship to solution structure due to their typically string-like conformations following activation and subsequent dissociation. Here, we used ion-ion chemistry to perform a broad survey of the gas-phase dissociation of charge-reduced protein complex ions, revealing general trends associated with the collisional ejection of compact, rather than unfolded, protein subunits. Furthermore, we also discover peptide and co-factor dissociation channels that dominate the product ion populations generated for such charge reduced complexes. We assess both sets of observations and discuss general principles that can be extended to the analysis of protein complex ions having unknown structures.

Ion mobility-mass spectrometry reveals conformational changes in charge reduced multiprotein complexes.

Characterizing intact multiprotein complexes in terms of both their mass and size by ion mobility-mass spectrometry is becoming an increasingly important tool for structural biology. Furthermore, the charge states of intact protein complexes can dramatically influence the information content of gas-phase measurements performed. Specifically, protein complex charge state has a demonstrated influence upon the conformation, mass resolution, ion mobility resolution, and dissociation properties of protein assemblies upon collisional activation. Here we present the first comparison of charge-reduced multiprotein complexes generated by solution additives and gas-phase ion-neutral reaction chemistry. While the charge reduction mechanism for both methods is undoubtedly similar, significant gas-phase activation of the complex is required to reduce the charge of the assemblies generated using the solution additive strategy employed here. This activation step can act to unfold intact protein complexes, making the data difficult to correlate with solution-phase structures and topologies. We use ion mobility-mass spectrometry to chart such conformational effects for a range of multi-protein complexes, and demonstrate that approaches to reduce charge based on ion-neutral reaction chemistry in the gas-phase consistently produce protein assemblies having compact, 'native-like' geometries while the same molecules added in solution generate significantly unfolded gas-phase complexes having identical charge states.