Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography?mass spectrometry

John, Harald; Walden, Michael; Schäfer, Sandra; Genz, Sandra; Forssmann, Wolf‐Georg

doi:10.1007/s00216-003-2298-y

Cited by 156 publications

(108 citation statements)

References 117 publications

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“…The improved robustness and sensitivity of LC-MSbased techniques have led to reliable alternatives for peptide quantification (21)(22)(23). We evaluated a method for the simultaneous quantification of ghrelin and desacyl-ghrelin that allows the monitoring of concentration-time profiles in biological matrices.…”

confidence: 99%

Simultaneous Quantification of Ghrelin and Desacyl-Ghrelin by Liquid Chromatography–Tandem Mass Spectrometry in Plasma, Serum, and Cell Supernatants

Rauh¹,

Gröschl²,

Rascher³

2007

Clinical Chemistry

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Background: A sensitive method specific for ghrelins is needed for investigations of this gastrointestinal peptide. Our aim was to develop and validate a quantitative mass spectrometry (MS) method to measure ghrelin and desacyl-ghrelin simultaneously. Methods: After deproteinization by precipitation, we performed reversed-phase separation with a rapid 2-column online extraction design coupled to a quadrupole mass spectrometer for electrospray ionization MS detection. Chromatography was performed on a C 18 monolithic column, with ammonium acetate buffer/methanol as the mobile phase and a chromatographic run time of 6 min/sample. The 4-fold-charged ions were used for multiple reaction monitoring experiments. Results: The method was linear with injections of 0.01-10 ng. Limits of detection and quantification were 0.02 and 0.07 g/L for ghrelin, respectively, and 0.03 and 0.35 g/L for desacyl-ghrelin. Intra-and interday imprecision (CVs) were 9%-4% and 12%-6% at concentrations of 0.33-5.93 g/L for ghrelin, respectively, and 16%-6% and 15%-8% at concentrations of 1.12-10.02 g/L for desacyl-ghrelin. The mean (SD) recoveries in plasma of added ghrelin and desacyl-ghrelin were 95.8% (12%) and 101% (1.2%), respectively. Using kinetic modeling, we determined the mean (SD) periods of halfchange (t 1/2 ) of ghrelin to be 156 (16) min in EDTA plasma and 49 (1) min in Li-heparin plasma. BlandAltman analysis showed that the median differences between EIA and liquid chromatography-tandem mass

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confidence: 99%

Simultaneous Quantification of Ghrelin and Desacyl-Ghrelin by Liquid Chromatography–Tandem Mass Spectrometry in Plasma, Serum, and Cell Supernatants

Rauh¹,

Gröschl²,

Rascher³

2007

Clinical Chemistry

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“…A LC-MS/MS-based absolute quantitative method (also known as AQUA), has been widely used in protein quantification in biological and clinical samples (John et al, 2004). However, the application for the measurement of membrane proteins has been delayed because of their hydrophobic nature and extremely low expression level (Barnidge et al, 2003).…”

mentioning

confidence: 99%

Absolute Difference of Hepatobiliary Transporter Multidrug Resistance-Associated Protein (MRP2/Mrp2) in Liver Tissues and Isolated Hepatocytes from Rat, Dog, Monkey, and Human

Li¹,

Zhang²,

Hua³

et al. 2008

Drug Metab Dispos

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ABSTRACT:We previously reported that hepatobiliary transporter multidrug resistance-associated protein (MRP2/Mrp2) is considered to be the major cause of the interspecies differences detected by efflux of fluorescent substrates in isolated hepatocytes. In the present study, the interspecies differences of MRP2/Mrp2 were first evaluated by quantitative real-time polymerase chain reaction and Western blotting. The mRNA levels were able to distinguish the difference among species with a rank order comparable with the corresponding activities observed, whereas the extents of the differences remained unknown. The cross-reactions of MRP2/Mrp2 protein of different species with anti-human MRP2 polyclonal antibody were found by Western blotting. However, because of the unknown binding affinity of antibody to MRP2/Mrp2 protein across species and lack of purified MRP2/Mrp2 proteins for calibration, the immunoblotting assay was excluded from the absolute quantification of MRP2/Mrp2 protein for multiple species. By using our newly developed liquid chromatography-tandem mass spectrometry quantification method, we were able to measure the absolute amount of MRP2/Mrp2 in liver tissues and isolated hepatocytes across species. Freshly isolated hepatocytes conserved MRP2/ Mrp2 protein levels that are comparable with those in the liver tissues. The amount of Mrp2 in rat liver was approximately 10-fold higher than that in other species. Moreover, a significant loss of Mrp2 protein in the membrane fraction of rat cryopreserved hepatocytes was observed. Thus, the absolute differences of MRP2/ Mrp2 levels in various species were determined, for the first time, by direct quantification. The results could potentially fill the translational gaps of in vitro/in vivo or preclinical species to human extrapolation of hepatobiliary elimination mediated by MRP2/ Mrp2.Xenobiotics and their metabolites are generally eliminated and detoxified by phase I and phase II enzymatic metabolism, by phase III transporter-mediated drug efflux to bile, or by both mechanisms. The excretion of drugs by hepatocytes into bile is one of the primary elimination routes for xenobiotics and the conjugate metabolites (Arias et al., 1993). In each stage of drug discovery, accurate prediction of human pharmacokinetics for a potential drug candidate is of great value (Mahmood, 1999). Even though the interspecies scaling methods based on physiologically allometric procedures have been successfully applied, particularly into extrapolation of hepatic enzymatic metabolism and urinary excretion (Dedrick et al., 1970;Iwatsubo et al., 1997;Ito et al., 1998), the in vitro or in vivo model for biliary excretion predication is far from being mature (Mahmood and Sahajwalla, 2002). The remarkable interspecies differences in biliary excretion of xenobiotics and drugs/metabolites (Ishizuka et al., 1999;Shilling et al., 2006) may cause significant overestimation of biliary excretion in humans simply by an exponential allometric extrapolation approach (Lave et al., 1999;Pahlman et al., ...

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“…Because of its specificity, MS is often preferred over immunochemical, radioactive, or optical methods for quantitative analyses, especially when the quantification is performed by liquid chromatography (LC)-MS͞MS and reaction monitoring, which completely eliminates the possibility of artifacts due to nonspecific signal (30). The demands of proteomics have resulted in the development of mass spectrometers of increasing sensitivity, with typical limits of detection in the subfemtomole range.…”

Section: Discussionmentioning

confidence: 99%

Ultrasensitive and absolute quantification of the phosphoinositide 3-kinase/Akt signal transduction pathway by mass spectrometry

Cutillas

Khwaja

Graupera

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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The phosphoinositide 3-kinase (PI3K)͞Akt pathway controls a vast array of normal physiological processes and is frequently aberrantly activated in cancer, thus identifying PI3K͞Akt-signaling components as promising drug targets in oncology. However, implementation of rational cancer therapies for this pathway needs robust and simple tools to stratify patients according to PI3K pathway activation and to validate and measure the impact of targeted inhibition on primary cancer tissues. Herein we present a technique for the quantification of the PI3K͞Akt-signaling pathway based on the mass spectrometric measurement of PI3K-dependent protein kinase activity in cell lysates. The concept of this application of MS is to exploit enzymatic activity to amplify the signal of the enzyme under study analogous to the PCR used to amplify nucleic acid sequences. We show that this approach allows quantitative analysis of a cell-signaling pathway with high sensitivity, precision of quantification, and specificity. Due to its special analytical capabilities and potential for multiplexing, this approach could contribute significantly to cell-signaling studies and to the development and implementation of personalized cancer therapies.quantitative analysis ͉ signaling pathway ͉ personalized therapies ͉ cancer stem cells ͉ patient stratification S ignal transduction pathways downstream of receptor tyrosine kinases control a large array of biological processes, including energy metabolism, cell proliferation, survival, cell motility, and immune responses. One of these pathways, the phosphoinositide 3-kinase (PI3K)͞Akt axis, is one of the most frequently deregulated pathways in cancer (1-4) by virtue of oncogenic mutation, by overexpression of its signaling components (5-8), by inactivation of the PTEN tumor-suppressor protein, which opposes PI3K action (9-12), or by being at the crossroads of upstream oncogenic signals (reviewed in ref. 4).Because of the importance of this pathway in the control of several biological and pathological processes (13-15), robust methods to quantify its activation are needed. PI3K catalyzes the phosphorylation of the lipid phosphatidylinositol-4,5-bisphosphate to form phosphatidylinositol-3,4,5-trisphosphate, a second messenger that is needed for the activation of downstream Ser͞Thr kinase protein kinases, such as Akt [also known as protein kinase B (PKB)] and phosphoinositide-dependent kinase-1. The assessment of lipid production by various approaches has turned out to be cumbersome, and it is thus far not possible to directly measure production of phosphatidylinositol-3,4,5-trisphosphate in tissue samples. Activation of the PI3K pathway is therefore commonly assessed by examination of signaling components downstream of PI3K. These components include Akt͞PKB and kinases farther downstream of the Akt͞PKB cascade, such as p70-S6K. Most often, this examination is performed by indirect assessment using immunochemical methods that measure the phosphorylation status of Akt on Ser-473 or Thr-308 or of S6, a ribos...

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Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography?mass spectrometry

Cited by 156 publications

References 117 publications

Simultaneous Quantification of Ghrelin and Desacyl-Ghrelin by Liquid Chromatography–Tandem Mass Spectrometry in Plasma, Serum, and Cell Supernatants

Simultaneous Quantification of Ghrelin and Desacyl-Ghrelin by Liquid Chromatography–Tandem Mass Spectrometry in Plasma, Serum, and Cell Supernatants

Absolute Difference of Hepatobiliary Transporter Multidrug Resistance-Associated Protein (MRP2/Mrp2) in Liver Tissues and Isolated Hepatocytes from Rat, Dog, Monkey, and Human

Ultrasensitive and absolute quantification of the phosphoinositide 3-kinase/Akt signal transduction pathway by mass spectrometry

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