Although LC-MS methods are increasingly used for the absolute quantification of proteins, the lack of appropriate internal standard (IS) hinders the development of rapid and standardized analytical methods for both in vitro and in vivo studies. Here, we have developed a novel method for the absolute quantification of a therapeutic protein, which is monoclonal antibody (mAb). The method combines liquid chromatography tandem mass spectrometry (LC-MS/MS) and protein cleavage isotope dilution mass spectrometry with the isotope-labeled mAb as IS. The latter was identical to the analyzed mAb with the exception that each threonine contains four (13)C atoms and one (15)N atom. Serum samples were spiked with IS prior to the overnight trypsin digestion and subsequent sample cleanup. Sample extracts were analyzed on a C18 ACE column (150 mm x 4.6 mm) using an LC gradient time of 11 min. Endogenous mAb concentrations were determined by calculating the peak height ratio of its signature peptide to the corresponding isotope-labeled peptide. The linear dynamic range was established between 5.00 and 1000 microg/mL mAb with accuracy and precision within +/-15% at all concentrations and below +/-20% at the LLOQ (lower limit of quantification). The overall method recovery in terms of mAb was 14%. The losses due to sample preparation (digestion and purification) were 72% from which about 32% was due to the first step of the method, the sample digestion. This huge loss during sample preparation strongly emphasizes the necessity to employ an IS right from the beginning. Our method was successfully applied to the mAb quantification in marmoset serum study samples, and the precision obtained on duplicate samples was, in most cases, below 20%. The comparison with enzyme-linked immunosorbent assay (ELISA) showed higher exposure in terms of AUC and Cmax with the LC-MS/MS method. Possible reasons for this discrepancy are discussed in this study. The results of this study indicate that our LC-MS/MS method is a simple, rapid, and precise approach for the therapeutic mAb quantification to support preclinical and clinical studies.

Two-dimensional (2-D) gel electrophoresis and peptide mass fingerprinting were used to investigate the natural variation in the proteome among 8 Arabidopsis thaliana ecotypes, of which 3 were previously shown to display atypical responses to environmental stress. Comparison of 2-D maps demonstrated that only one-quarter of spots was shared by all accessions. On the other hand, only 15% of the 25 majors spots accounting for half the total protein amount could be classified as major spots in all ecotypes. Identification of these major spots demonstrated large differences between the major functions detected. Accordingly, the proteomes appeared to reveal important variations in terms of function between ecotypes. Hierarchical clustering of proteomes according to either the amount of all anonymous spots, that of the 25 major spots or the functions of these major spots identified the same classes of ecotypes, and grouped the three atypical ecotypes. It is proposed that proteome comparison has the capacity to evidence differences in the physiological status of ecotypes. Results are discussed with respect to the possibility to infer such differences from limited comparisons of major proteins. It is concluded that classical proteomics could constitute a powerful tool to mine the biodiversity between ecotypes of a single plant species.

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In the present study, the application of a liquid chromatography high-resolution mass spectrometry (LC-HRMS) analytical assay for the quantitative analysis of a recombinant human immunoglobulin G1 (hIgG1) in rat serum is reported using three generic peptides GPSVFPLAPSSK (GPS), TTPPVLDSDGSFFLYSK (TTP), and VVSVLTVLHQDWLNGK (VVS). Moreover, the deamidation site of a fourth peptide FNWYVDGVEVHNAK (FNW) was identified and further excluded from the assay evaluation due to the inaccuracy of the quantitative results. The rat serum samples were spiked with a fully labeled hIgG1 as internal standard (ISTD). The digestion with trypsin was performed onto the pellet prior to peptide analysis by LC-HRMS using a quadrupole time of flight (QTOF) mass analyzer operating in selected reaction monitoring (SRM) mode with enhanced duty cycles (EDC). The assay linearity for the three investigated peptides was established for a hIgG1 (hIgG1A) from 1.00 to 1000 μg mL(-1) with a mean coefficient of determination (R (2)) higher than 0.9868. The inter-day accuracy and precision obtained in rat serum over 3 days were ≤11.4 and ≤10.5%, respectively. Short-term stability on the auto-sampler at 6 °C for 30 h, at RT for 48 h, and a 100-fold dilution factor were demonstrated. In addition, QC samples prepared in cynomolgus monkey serum and measured with the present method met the acceptance criteria of ±20.0 and ≤20.0% for all three peptides regarding accuracy and precision, respectively. The LC-HRMS method was applied to the analysis of samples from five individual cynomolgus monkeys dosed with a second hIgG1 (hIgG1B) and consistent data were obtained compared to the LC-MS/MS method (conventional triple quadrupole (QqQ) mass analyzer operating in SRM). The present data demonstrate that LC-HRMS can be used for the quantitative analysis of hIgG1 in both species and that quantification is not only limited to classical QqQ instruments.

A new class of 4-aminoquinolines was synthesized and evaluated in vitro for antiplasmodial activity against both the chloroquine-sensitive (3D7) and -resistant (K1 and W2) strains. The most active compounds 3c−3e had acceptable cytotoxicity but showed strong inhibition toward a panel of cytochrome P450 enzymes in vitro. Pharmacokinetic studies on 3d and 3e in mice showed that they had moderate half-life (4−6 h) and low oral bioavailability. The front runner compound 3d exhibited moderate inhibition of the malaria parasite on P. berghei infected mice following oral administration (5 mg/kg), achieving reduction of parasitemia population by 47% on day 7.