Ion mobility and mass spectrometry techniques, combined with electrospray ionization, have been used to examine distributions of poly(ethylene glycols) (PEG) with average molecular masses of 6550 and 17900 Da. The analysis provides information about the polymer size distributions as well as smaller oligomers existing over a wide range of charge states and sizes (i.e., [HO(CH2CH2O)xH + nCs]n+, where x ranges from 21 to 151 and n = 2 to 11 for the 6550 Da sample; and, x ranges from 21 to 362 and n = 2 to 23 for the 17 900 Da sample). The present data show that oligomer distributions also fall into families, corresponding to much narrower size distributions for individual charge states; this dramatically simplifies data analysis. For example, we show evidence for baseline resolution of the +10 charge state of polymers. Unlike the charge-state trends reported previously for peptide ion families, which show generally increasing mobilities with increasing charge state (for a given m/z value), the mobilities of [HO(CH2CH2O)xH + nCs]n+ families generally decrease with increasing charge state. This requires that the addition of charges leads to substantial changes in the average structures of the ions. Comparisons of cross section calculations from molecular modeling results for multiply cesiated PEG ions with experimental cross sections indicate that these ions adopt highly extended (in many cases nearly linear) conformations, except for the high degree of coordination of the charged sites.

Aberrant glycosylation of human glycoproteins is related to various physiological states, including the onset of diseases such as cancer. Consequently, the search for glycans that could be markers of diseases or targets of therapeutic drugs has been intensive. Here, we describe a high-throughput ion mobility spectrometry/mass spectrometry analysis of N-linked glycans from human serum. Distributions of glycans are assigned according to their m/z values, while ion mobility distributions provide information about glycan conformational and isomeric composition. Statistical analysis of data from 22 apparently healthy control patients and 39 individuals with known diseases (20 with cirrhosis of the liver and 19 with liver cancer) shows that ion mobility distributions for individual m/z ions appear to be sufficient to distinguish patients with liver cancer or cirrhosis. Measurements of glycan conformational and isomeric distributions by IMS-MS may provide insight that is valuable for detecting and characterizing disease states.

The increasing prevalence of drug-resistant tuberculosis highlights the need for identifying new antitubercular drugs that can treat these infections. The antigen 85 (Ag85) complex has emerged as an intriguing mycobacterial drug target due to its central role in synthesizing major components of the inner and outer leaflets of the mycobacterial outer membrane. Here we identify ebselen as a potent inhibitor of the Mycobacterium tuberculosis Ag85 complex. Mass spectrometry data show that ebselen binds covalently to a cysteine residue (C209) located near the Ag85C active site. The crystal structure of Ag85C in the presence of ebselen shows that C209 modification restructures the active site, thereby disrupting the hydrogen-bonded network within the active site that is essential for enzymatic activity. C209 mutations display marked decreases in enzymatic activity. These data suggest that compounds using this mechanism of action will strongly inhibit the Ag85 complex and minimize the selection of drug resistance.

Ion© mobility-mass© spectrometry© (IMS-MS)© and© molecular© modeling© techniques© have© been© used to©characterize©ovalbumin©N-linked©glycans.©Some©glycans©from©this©glycoprotein©exist©as multiple© isomeric© forms.© The© gas-phase© separation© makes© it© possible© to© resolve© some© isomers before© MS© analysis.© Comparisons© of© experimental© cross© sections© for© selected© glycan© isomers with©values©that©are©calculated©for©iterative©structures©generated©by©molecular©modeling techniques© allow© the© assignment© of© sharp© features© to© specific© isomers.© We© focus© here© on© an example© glycan© set,© each© having© a© m/z value© of© 1046.52© with© formula© [H 5 N 4© ϩ© 2Na] 2ϩ ,© where H© corresponds© to© a© hexose,© and© N© to© a© N-acetylglucosamine.© This© glycan© appears© to© exist© as three© different© isomeric© forms© that© are© assignable© based© on© comparisons© of© measured© and calculated© cross© sections.© We© estimate© the© relative© ratios© of© the© abundances© of© the© three© isomers to© be© in© the© range© of© ϳ1.0:1.35:0.85© to© ϳ1.0:1.5:0.80.© In© total,© IMS-MS© analysis© of© ovalbumin N-linked©glycans©provides©evidence©for©19©different©glycan©structures©corresponding©to high-mannose© and© hybrid© type© carbohydrates© with© a© total© of© 42© distinct© features© related© to isomers© and/or© conformers

No abstract

The conformation of an elastin-mimetic recombinant protein, [(VPGVG)4(VPGKG)]39, is investigated using solid-state NMR spectroscopy. The protein is extensively labeled with 13C and 15N, and two-dimensional 13C-13C and 15N-13C correlation experiments were carried out to resolve and assign the isotropic chemical shifts of the various sites. The Pro 15N, 13Calpha, and 13Cbeta isotropic shifts, and the Gly-3 Calpha isotropic and anisotropic chemical shifts support the predominance of type-II beta-turn structure at the Pro-Gly pair but reject a type-I beta-turn. The Val-1 preceding Pro adopts mostly beta-sheet torsion angles, while the Val-4 chemical shifts are intermediate between those of helix and sheet. The protein exhibits a significant conformational distribution, shown by the broad line widths of the 15N and 13C spectra. The average chemical shifts of the solid protein are similar to the values in solution, suggesting that the low-hydration polypeptide maintains the same conformation as in solution. The ability to measure these conformational restraints by solid-state NMR opens the possibility of determining the detailed structure of this class of fibrous proteins through torsion angles and distances.

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 μM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 μM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure–activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines.

Altered branching and aberrant expression of N-linked glycans is known to be associated with disease states such as cancer. However, the complexity of determining such variations hinders the development of specific glycomic approaches for assessing disease states. Here, we examine a combination of ion mobility spectrometry (IMS) and mass spectrometry (MS) measurements, with principal component analysis (PCA) for characterizing serum N-linked glycans from 81 individuals: 28 with cirrhosis of the liver; 25 with liver cancer; and 28 apparently healthy. Supervised PCA of combined ion-mobility profiles for several, to as many as ten different mass-to-charge ratios for glycan ions, improves the delineation of diseased states. This extends an earlier study [J.Proteome Res. 2008, 7, 1109-1117] of isomers associated with a single glycan (S1H5N4) in which PCA analysis of the IMS profiles appeared to differentiate the liver cancer group from the other samples. Although performed on a limited number of test subjects, the combination of IMS-MS for different combinations of ions and multivariate PCA analysis shows promise for characterizing disease states.