Chemical, Analytical and Pharmacokinetic Characterisation of RO7304898, an API Consisting of Two Rapidly Interconverting Diastereoisomers.

PURPOSE: Exploration of the chemical, analytical and pharmacokinetic properties of the API, RO7304898, an allosteric EGFR inhibitor, intended to be developed as a mixture of two rapidly interconverting diastereoisomers with composition ratio of approximately 1:1. METHODS: Assessment of diastereoisomer stereochemistry, interconversion rates, binding to EGFR protein, metabolic stability and in vivo PK in Wistar-Han rats was conducted. RESULTS: The two diastereoisomers of the API undergo fast interconversion at physiologically relevant pH and direct EGFR binding studies revealed diastereoisomer B to be the active moiety. Pharmacokinetic studies in rat revealed a low-moderate total plasma clearance of the API along with similar plasma concentration-time profiles for diastereoisomers A and B, and the diastereoisomeric ratio reached stable equilibrium favoring formation of the potent diastereoisomer B. In in vitro incubations, the API was metabolically stable in plasma and hepatocyte suspension incubations in all species tested except that of rat hepatocytes. Additionally, only small species differences in the A:B composition were observed in vitro with the potent diastereoisomer B being the predominant form. CONCLUSIONS: We demonstrated that the API, a mixture of two diastereoisomers; A (impotent) and B (potent), undergoes rapid interconversion which is faster than the apparent distribution and elimination rates of the individual diastereoisomers in vivo in rat, serving to diminish concerns that separate diastereoisomer effects may occur in subsequent pharmacologic and pivotal toxicological studies. Whilst vigilant monitoring of the diastereoisomeric ratio will need to be continued, this data adds confidence on the development pathway for this API to the clinic.

Publisher Correction: OpenSWATH enables automated, targeted analysis of data-independent acquisition MS data.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen-treated three-dimensional liver microtissues.

The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP)(1)-treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD). Our findings imply that DIA should be the preferred method for quantitative protein profiling.

In-spray supercharging of peptides and proteins in electrospray ionization mass spectrometry.

Enhanced charging, or supercharging, of analytes in electrospray ionization mass spectrometry (ESI MS) facilitates high resolution MS by reducing an ion mass-to-charge (m/z) ratio, increasing tandem mass spectrometry (MS/MS) efficiency. ESI MS supercharging is usually achieved by adding a supercharging reagent to the electrospray solution. Addition of these supercharging reagents to the mobile phase in liquid chromatography (LC)-MS/MS increases the average charge of enzymatically derived peptides and improves peptide and protein identification in large-scale bottom-up proteomics applications but disrupts chromatographic separation. Here, we demonstrate the average charge state of selected peptides and proteins increases by introducing the supercharging reagents directly into the ESI Taylor cone (in-spray supercharging) using a dual-sprayer ESI microchip. The results are comparable to those obtained by the addition of supercharging reagents directly into the analyte solution or LC mobile phase. Therefore, supercharging reaction can be accomplished on a time-scale of ion liberation from a droplet in the ESI ion source.

On the utility of isotopic fine structure mass spectrometry in protein identification.

Modern mass spectrometry (MS)-based protein identification and characterization relies upon accurate mass measurements of the (13)C isotopic distributions of the enzymatically produced peptides. Interestingly, obtaining peptide elemental composition information from its isotopic fine structure mass spectrum to increase the confidence in peptide and protein identification has not yet been developed into a bottom-up proteomics-grade analytical approach. Here, we discuss the possible utility and limitations of the isotopic fine structure MS for peptide and protein identification. First, we in silico identify the peptides from the E. coli tryptic digest and show the increased confidence in peptide identification by consideration of the isotopic fine structures of these peptides as a function of mass and abundance accuracies. In the following, we demonstrate that the state-of-the-art high magnetic field Fourier transform ion cyclotron resonance (FT-ICR) MS allows a routine acquisition of the isotopic fine structure information of a number of isobaric peptide pairs, including a pair of peptides originating from E. coli. Finally, we address the practical limitation of the isotopic fine structure MS implementation in the time-constraint experiments by applying an advanced signal processing technique, filter diagonalization method, to the experimental transients to overcome the resolution barrier set by the typically applied Fourier transformation. We thus demonstrate that the isotopic fine structures of peptides may indeed improve the peptide and possibly protein identification, can be produced in a routine experiment by the state-of-the-art high resolution mass spectrometers, and can be potentially obtained on a chromatographic time-scale of a typical bottom-up proteomics experiment. The latter one requires at least an order of magnitude increase in sensitivity of ion detection, which presumably can be realized using high-field Orbitrap FTMS and/or future generation of ultrahigh magnetic field FT-ICR MS equipped with harmonized ICR cells.

High-resolution and tandem mass spectrometry--the indispensable tools of the XXI century.

Mass spectrometry-based qualitative and quantitative (bio)molecular analysis is a corner stone in the state-of-the-art pipelines in systems biology and environmental sciences. High-resolution and efficient tandem mass spectrometry methods and techniques are the essential analytical capabilities for the in-depth analysis of extremely complex mixtures of (bio)molecules of a very broad dynamic range of concentrations. Here, we briefly review the advantages and limitations of the current mass spectrometry with a focus on resolution, or resolving power, and methods of (bio)molecular fragmentation in the gas phase. We conclude with an outlook that considers possible avenues for further mass spectrometry-based method and technique development, indispensable for advancing the challenging real-life mass spectrometry applications in the XXI century.

Growth mechanisms involved in the synthesis of smooth and microtextured films by acetylene magnetron discharges.

The growth of hydrogenated amorphous carbons (a-C:H) produced by continuous or pulsed discharges of acetylene (C(2)H(2)) in an unbalanced magnetron setup was investigated. At 5 × 10(-3) Torr, only smooth films are obtained, whereas at 5 × 10(-1) Torr using a pulsed discharge some microtextured films are formed if the duty cycle is low. The morphology of these microtextured films consists of nanoparticles, filamentary particles, and particular agglomerates ("microflowers"). This paper presents a study of acetylene gas phase polymerization by mass spectrometry, and a detailed analysis of bulk structure of films by combining three techniques which include IR spectroscopy, Raman spectroscopy, and laser desorption/ionization Fourier transform mass spectrometry (LDI-FTMS). Finally, based on the study of gas phase and film structure, we propose a model for the growth of both smooth and microtextured films.

Laser desorption/ionization Fourier transform mass spectrometry of thin films deposited on silicon by plasma polymerization of acetylene.

Thin films deposited on silicon substrate by three different methods of plasma polymerization of acetylene were analyzed by direct laser desorption/ionization Fourier transform mass spectrometry. High-resolution mass spectra showed the presence of carbon clusters and hydrocarbon oligomers in different relative abundances. During unipolar and continuous discharge polymerization of acetylene-hydrogen gas mixtures, quadrupole mass spectra of the plasma constituents showed the presence of molecular species with m/z lower than 100--mainly peaks of C(4)H(2) and C(6)H(2). Films produced had smooth surfaces and the corresponding LDI-FTMS spectra displayed only carbon cluster signals in the positive ion mode and both hydrocarbon and carbon cluster signals (with much higher relative abundance of carbon cluster signals) in the negative ion mode. Alternatively, during bipolar discharge with either higher acetylene gas flux (>40 cm(3)/min) or longer deposition times (>10 min), quadrupole mass spectra of the plasma constituents showed signals corresponding to polycyclic aromatic hydrocarbons (PAH) with m/z higher than 100. SEM pictures of the bipolar thin films demonstrated the presence of "flower" structures and nanoparticles developed on the surface. LDI-FTMS spectra of such thin films showed either total absence or lower relative abundance of carbon cluster signals, compared with hydrocarbon signals.

Wide mass range trapping using a 7-T internal source matrix-assisted laser desorption/ionization Fourier transform mass spectrometer.

A wide mass range trapping experiment using internal source matrix-assisted laser desorption-Fourier transform mass spectrometry (MALDI-FTMS) was evaluated. In this method, the front trap plate potential is ramped up and the rear trap plate potential is simultaneously decreased using a cubic cell to trap ions over a wide range of mass-to-charge ratios. To apply this to MS/MS experiments, a second ion ejection procedure would remove unwanted ions, with the selected remaining ions then fragmented by collision-induced dissociation. In measurements using a 7.2-T unshielded magnet presented here, an approximately equimolar mixture of a set of poly(ethylene glycol) (PEG) species for the ramped measurements had peak areas of 1.0:1.0:1.0:1.0, as did the previously described integral method which gave peak areas of 1.0:1.1:1.0:1.0, in good agreement with the known composition of the samples deposited on the MALDI probe tip. Comparative MALDI-TOF in reflectron mode results were of similar quality for the equimolar mixture, giving a ratio of 1.0:1.0:1.2:0.9. All methods failed to varying degrees when individual PEG compositions of the trial mixture were changed. However, the previously described integral method showed relatively better results for all but the PEG 8000 doubled mixture.