Understanding the effect of the HCV polymerase inhibitor mericitabine on early viral kinetics in the phase 2 JUMP-C and PROPEL studies.

AIMS: The aim was to evaluate early viral kinetics in patients receiving mericitabine [hepatitis C virus (HCV) nucleoside polymerase inhibitor] with peginterferon alfa-2a (40KD) and ribavirin in two clinical trials (PROPEL and JUMP-C). METHODS: We examined rapid virological responses (RVRs; week 4 HCV RNA <15 IU ml(-1) ) and complete early virological responses (cEVR; week 12 HCV RNA <15 IU ml(-1) ) in HCV genotype 1/4-infected patients receiving mericitabine (500 or 1000 mg) or placebo twice daily plus peginterferon alfa-2a and ribavirin. RESULTS: Among IL28B rs12979860 CC genotype patients receiving 500 or 1000 mg mericitabine or placebo, respectively, RVR rates were 64.3% (95% confidence interval: 38.8-83.7%), 95.1% (83.9-98.7%) and 33.3% (20.2-49.7%), and cEVR rates were 100% (78.5-100%), 100% (91.4-100%) and 80.6% (65.0-90.3%). Among non-CC genotype patients, RVR rates were 26.5% (14.6-43.1%), 52.3% (43.0-61.3%) and 5.7% (2.2-13.8%), and cEVR rates were 76.5% (60.0-87.6%), 84.6% (76.6-90.1%) and 28.6% (19.3-40.1%), respectively. In multiple regression analysis, IL28B genotype (P < 0.0001), mericitabine dose (P < 0.0001) and bodyweight (P = 0.0009) were associated with first-phase (α) slope (change in log10 HCV RNA from baseline to week 1). CONCLUSIONS: Mericitabine-containing triple therapy reduces the impact of IL28B genotype on RVR and cEVR compared with peginterferon alfa-2a and ribavirin dual therapy. The IL28B genotype, mericitabine dose and bodyweight are the most important factors associated with the α slope, and there is no evidence of a pharmacokinetic drug-drug interaction between mericitabine and ribavirin.

A linear ion trap mass spectrometer with versatile control and data acquisition for ion/ion reactions.

A linear ion trap (LIT) with electrospray ionization (ESI) for top-down protein analysis has been constructed. An independent atmospheric sampling glow discharge ionization (ASGDI) source produces reagent ions for ion/ion reactions. The device is also meant to enable a wide variety of ion/ion reaction studies. To reduce the instrument's complexity and make it available for wide dissemination, only a few simple electronics components were custom built. The instrument functions as both a reaction vessel for gas-phase ion/ion reactions and a mass spectrometer using mass-selective axial ejection. Initial results demonstrate trapping efficiency of 70% to 90% and the ability to perform proton transfer reactions on intact protein ions, including dual polarity storage reactions, transmission mode reactions, and ion parking.

Evidence for unfolding and refolding of gas-phase cytochrome C ions in a Paul trap.

The folding pathways of gas-phase cytochrome c ions produced by electrospray ionization have been studied by an ion trapping/ion mobility technique that allows conformations to be examined over extended timescales (10 ms to 10 s). The results show that the +9 charge state emerges from solution as a compact structure and then rapidly unfolds into several substantially more open structures, a transition that requires 30-60 ms; over substantially longer timescales (250 ms to 10 s) elongated states appear to refold into an array of folded structures. The new folded states are less compact than those that are apparent during the initial unfolding. Apparently, unfolding to highly open conformations is a key step that must occur before +9 ions can sample more compact states that are stable at longer times.

Dissociation of different conformations of ubiquitin ions.

The fragmentation pathways of different conformations of three charge states of ubiquitin ions are examined using ion mobility/collisional activation/time-of-flight techniques. Mass spectra for fragments for different conformers of a single charge state appear to be identical (within the experimental reproducibility). These results are consistent with a mechanism in which different conformers of each charge state rearrange to similar dissociation transition states prior to fragment formation.

Accelerating high quality bioanalytical LC/MS/MS assays using fused-core columns.

High quality, ultra-fast bioanalytical LC/MS/MS methods were developed using short columns packed with fused-core particles and high (1.0-3.0 mL/min) flow rates. For more than two years, at flow rates up to 3.0 mL/min, using 0.33 min non-ballistic gradients, these methods were shown to provide comparable or better performance than slower assays for accuracy, precision, sensitivity, specificity, and ruggedness, and met all criteria required by the bioanalytical regulatory guidance.

Effects of ion/ion proton transfer reactions on conformation of gas-phase cytochrome c ions.

Positive ions from cytochrome c are studied in a 3-D ion trap/ion mobility (IM)/quadrupole-time-of-flight (TOF) instrument with three independent ion sources. The IM separation allows measurement of the cross section of the ions. Ion/ion reactions in the 3-D ion trap that remove protons cause the cytochrome c ions to refold gently without other degradation of protein structure, i.e., fragmentation or loss of heme group or metal ion. The conformation(s) of the product ions generated by ion/ion reactions in a given charge state are similar regardless of whether the cytochrome c ions are originally in +8 or +9 charge states. In the lower charge states (+1 to +5) cytochrome c ions made by the ion/ion reaction yield a single IM peak with cross section of approximately 1110 to 1180 A(2), even if the original +8 ion started with multiple conformations. The conformation expands slightly when the charge state is reduced from +5 to +1. For product ions in the +6 to +8 charge states, ions created from higher charge states (+9 to +16) by ion/ion reaction produce more compact conformation(s) in somewhat higher abundances compared with those produced directly by the electrospray ionization (ESI) source. For ions in intermediate charge states that have a variety of resolvable conformers, the voltage used to inject the ions into the drift tube, and the voltage and duration of the pulse that extracts ions from the ion trap, can affect the observed abundances of various conformers.

An ion trap-ion mobility-time of flight mass spectrometer with three ion sources for ion/ion reactions.

This instrument combines the capabilities of ion/ion reactions with ion mobility (IM) and time-of-flight (TOF) measurements for conformation studies and top-down analysis of large biomolecules. Ubiquitin ions from either of two electrospray ionization (ESI) sources are stored in a three dimensional (3D) ion trap (IT) and reacted with negative ions from atmospheric sampling glow discharge ionization (ASGDI). The proton transfer reaction products are then separated by IM and analyzed via a TOF mass analyzer. In this way, ubiquitin +7 ions are converted to lower charge states down to +1; the ions in lower charge states tend to be in compact conformations with cross sections down to approximately 880 A(2). The duration and magnitude of the ion ejection pulse on the IT exit and the entrance voltage on the IM drift tube can affect the measured distribution of conformers for ubiquitin +7 and +6. Alternatively, protein ions are fragmented by collision-induced dissociation (CID) in the IT, followed by ion/ion reactions to reduce the charge states of the CID product ions, thus simplifying assignment of charge states and fragments using the mobility-resolved tandem mass spectrum. Instrument characteristics and the use of a new ion trap controller and software modifications to control the entire instrument are described.

A quadrupole ion trap mass spectrometer with three independent ion sources for the study of gas-phase ion/ion reactions.

An instrument for the study of gas-phase ion/ion reactions in which three independent sources of ions, namely, two electrospray ionization sources and one atmospheric sampling glow discharge ionization source, are interfaced to a quadrupole ion trap mass analyzer is described. This instrument expands the scope of gas-phase ion/ion reaction studies by allowing for manipulation of the charge states of multiply charged reactant and product ions. Examples are provided involving the formation of protein-protein complexes in the gas phase. Complexes with charge states that cannot be formed from reactant ion charge states present in the normal electrospray charge state distributions can be formed in the new apparatus. Strategies that rely on both reactant ion charge state manipulation and product ion charge state manipulation are demonstrated. In addition, simplification of product ion spectra generated from dissociation of complexes formed via ion/ion reactions can be effected by using the discharge source to reduce the charge state of the product ions to primarily 1+.

Gas-phase separations of protein and peptide ion fragments generated by collision-induced dissociation in an ion trap.

Ion mobility/time-of-flight mass spectrometry techniques have been used to examine distributions of fragment ions generated by collision-induced dissociation (CID) in a quadrupole ion trap. The mobility-based separation step prior to mass-to-charge (m/z) analysis reduces spectral congestion and provides information that complements m/z-based assignments of peaks. The approach is demonstrated by examining fragmentation patterns of insulin chain B (a 30-residue peptide), and ubiquitin (a protein containing 76 amino acids). Some fragments of ubiquitin show evidence for multiple stable conformations.