Ethylenediaminetetraacetic acid increases identification rate of phosphoproteomics in real biological samples.

We have developed a novel approach to enhance phosphopeptide identification in liquid chromatography/mass spectrometry (LC/MS)-based phosphoproteomics. After enrichment of phosphopeptides with immobilized metal affinity chromatography (IMAC) and titanium dioxide (TiO(2)) microcolumns, samples were coinjected with ethylenediaminetetraacetic acid (EDTA) into LC/MS. This procedure decreased the MS peak intensity of nonphosphorylated peptides, but not that of phosphopeptides, and as a result, the number of identified phosphopeptides was increased. EDTA appeared to have no effect on liquid chromatographic separation of phosphorylated and nonphosphorylated peptides. Although the mechanism of the positive effect of EDTA on identification of phosphopeptides is unknown, and we have never observed metal ion adduct peaks in LC/MS spectra, coinjection of EDTA seemed to enhance phosphopeptide recovery from the LC/MS system. This simple technique was successfully applied to the identification of phosphopeptides in mouse brain (2938 phosphopeptides), human plasma (127 phosphopetides), and human cerebrospinal fluid (CSF) (123 phosphopeptides). We also identified nonphosphopeptides in the same samples using a two-dimensional (2D) LC/MS-based shotgun approach. The results overall indicated that 20-25% of brain proteins were phosphorylated, while only 1-2% of proteins in plasma and CSF were phosphorylated. These ratios were almost constant throughout the range of protein expression levels. In addition, EDTA-enhanced phosphoproteomics could identify low-abundance proteins in the samples, because nonphosphoproteins corresponding to more than one-third of the identified phosphoproteins could not be identified by 2D-LC/MS. Finally, we were able to find that the newly developed approach was very effective for the phosphoproteome analysis in Alzheimer disease model mice brain.

Polar anionic metabolome analysis by nano-LC/MS with a metal chelating agent.

We have developed a practical method for the comprehensive analysis of polar anionic metabolites in biological samples with the use of a nano-LC/MS system. A polyamine-bonded polymer-based apHera NH2 column, which is compatible with ammonium carbonate buffer, effectively retained anionic polar metabolites, such as organic acids, sulfates, and phosphates, but multiply phosphorylated or carboxylated compounds showed highly distorted peak shapes on chromatograms. We found that addition of a trace amount of the metal chelating reagent ethylenediaminetetraacetic acid (EDTA) to the sample solution dramatically improved peak shapes of multiply charged anionic compounds, even though the mass spectra showed no trace of adduct ions in the absence of EDTA. The detection limits of typical polar anionic metabolites in the full-scan mode were from 0.19 to 2.81 pmol. After optimization of all the procedures from sample preparation to nano-LC/MS analysis, we applied our method to real biological samples: Hela cells, mouse brain, human cerebrospinal fluid (CSF), and human plasma. Our results indicated that phosphorylated metabolites were abundant in Hela cells and brain, while plasma and cerebrospinal fluid (CSF) mostly contained organic acids. Phosphorylated compounds might not be secreted into CSF/plasma or might be unstable in CSF/plasma. Finally, the method was used to examine the mode of action of the anticancer drug methotrexate (MTX), which inhibits purine de novo biosynthesis and thymidine biosynthesis. In addition of the expected changes of metabolite levels, we found that a previously unreported metabolite, probably a methylated uridine 5'-triphosphate (UTP), was produced by MTX-treated Hela cells.

Quantitative profiling of polar cationic metabolites in human cerebrospinal fluid by reversed-phase nanoliquid chromatography/mass spectrometry.

Reversed-phase (RP) nanoliquid chromatography (LC)/mass spectrometry (MS) is widely used for proteome analysis, but hydrophilic metabolites are poorly retained on RP columns. We describe here the development and application of an efficient, robust, and quantitative nano-LC/MS method for cationic metabolome analysis in the positive ionization mode without any derivatization of analytes. Various stationary phases for nano-LC, coating of the internal wall of the capillary column, and various mobile phases were evaluated in terms of separation and peak shapes for 33 hydrophilic metabolites, including nonderivatized amino acids. Polar cationic compounds were strongly bound to mixed-functional RP with cation exchange mode resin, and the best separation was obtained with hydrophilic internal wall coating and a two-step trifluoroacetic acid (TFA) gradient in methanol as the mobile phase. Simple, but optimized, sample processing and the use of a high content of methanol allowed robust nano-LC/MS analysis. Our developed method was applied for biomarker discovery in Alzheimer's disease (AD). Several hundred peaks were detected from 10 microL of cerebrospinal fluid (CSF). In a principal component analysis (PCA) plot using peak intensities without normalization, peak separation depended on the experimental date, not disease state. Therefore, constant amounts of two stable isotope-labeled amino acids, Val and Lys, were added as internal standards (ISs) to each sample before processing. These ISs were eluted in different gradient slopes in the two-step gradient, and the normalized peak ratios using the corresponding ISs gave a unique group of PCA scores which could distinguish AD CSF samples from age-matched control CSF samples.

LC/ESI-tandem mass spectrometric determination of bile acid 3-sulfates in human urine 3beta-Sulfooxy-12alpha-hydroxy-5beta-cholanoic acid is an abundant nonamidated sulfate.

We developed a highly sensitive and quantitative method to detect bile acid 3-sulfates in human urine employing liquid chromatography/electrospray ionization-tandem mass spectrometry. This method allows simultaneous analysis of bile acid 3-sulfates, including nonamidated, glycine-, and taurine-conjugated bile acids, cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), and lithocholic acid (LCA), using selected reaction monitoring (SRM) analysis. The method was applied to analyze bile acid 3-sulfates in human urine from healthy volunteers. The results indicated an unknown compound with the nonamidated common bile acid 3-sulfates on the chromatogram obtained by the selected reaction monitoring analysis. By comparison of the retention behavior and MS/MS spectrum of the unknown peak with the authentic specimen, the unknown compound was identified as 3beta,12alpha-dihydroxy-5beta-cholanoic acid 3-sulfate.

A phase I, dose-escalation study of the multitargeted receptor tyrosine kinase inhibitor, golvatinib, in patients with advanced solid tumors.

PURPOSE: Receptor tyrosine kinases c-Met and Ron transduce signals regulating cell migration and matrix invasion. This phase I dose-escalation trial tested golvatinib, a highly potent, small-molecule, ATP-competitive inhibitor of c-Met and multiple members of the Eph receptor family plus c-Kit and Ron. EXPERIMENTAL DESIGN: Patients with advanced solid tumors received golvatinib orally, once daily, continuously. Using a "3+3" design, dosing started at 100 mg once daily, escalating to the maximum tolerated dose (MTD) defined by dose-limiting toxicities. Pharmacokinetic, pharmacodynamic, and preliminary antitumor activity was assessed during dose escalation and in a MTD expansion cohort. RESULTS: Thirty-four patients were treated at six dose levels. The MTD was determined as 400 mg once daily. Three dose-limiting toxicities were observed: grade 3 increased γ-glutamyltransferase and alkaline phosphatase (200 mg), repeated grade 2 fatigue, and grade 3 fatigue (50.0%). Frequent treatment-related adverse events (with incidence >10%) included diarrhea (58.8%), nausea (50%), vomiting (44.1%), fatigue (41.2%), decreased appetite (32.4%), elevated alanine aminotransferase (32.4%), elevated aspartate aminotransferase (20.6%), dry skin (11.8%), and dysgeusia (11.8%). Best overall response was stable disease (median duration 85 days, range 85-237). Pharmacokinetics demonstrated high variability, although maximum plasma concentration and area under the plasma concentration-time curve increased with dose. Soluble urokinase-type plasminogen activator receptor, VEGFR2, c-Met, and angiopoietin-2 levels increased after dose. Posttreatment decrease in either p-c-Met or p-ERK was observed in 3 of 4 paired biopsies at MTD. CONCLUSIONS: Golvatinib at the MTD of 400 mg once daily was well tolerated with pharmacodynamic evidence of c-Met target modulation.

Practical metabolomics in drug discovery.

IMPORTANCE OF THE FIELD: Metabolomics is increasingly becoming an important field in the pharmaceutical industry to support the discovery and development of therapeutic agents. It allows the comprehensive and simultaneous profiling of hundreds of discrete biologically important molecules, including amino acids, sugars, lipids and exogenous substances from biological fluids and tissues. Metabolomics is the 'omics' field that most represents the interplay of internal biological regulation and external environmental influences on disease, thereby being of particular importance to disease mitigation and management. AREAS COVERED IN THIS REVIEW: Technological advances in the experimental work flow, analytical detection strategies and bioinformatics tools have enabled metabolomics studies to become increasingly comprehensive, robust and informative for the understanding of disease, drug action and the development of biomarkers. This review will focus on the practical aspects of metabolomics studies as they have been applied to the study of mammalian biological systems, specifically targeted to the steps of experimental design with regard to sample preparation, sample analysis and data analysis of both polar and non-polar metabolites. WHAT THE READER WILL GAIN: The reader will gain an overview of the field of metabolomics as it applies to drug development and the practical issues involved with experimental design. We will discuss the various methods of sample preparation and analysis as they apply to different classes of metabolites and highlight recent advances in the field that illustrate these methods. TAKE HOME MESSAGE: The field of metabolomics is a rapidly expanding discipline that is being applied to various aspects of drug development. The large diversity of metabolites found in nature dictates that different methods be developed for the investigation of different classes of metabolites. As the field of metabolomics continues to mature, it is likely that it will play an increasingly important role in the characterization of disease and the future development of biomarkers to assess drug efficacy and safety.