Identification of Plasma Membrane Glycoproteins Specific to Human Glioblastoma Multiforme Cells Using Lectin Arrays and LC‐MS/MS

Glioblastoma, also known as glioblastoma multiforme (GBM), is the most malignant type of brain cancer and has poor prognosis with a median survival of less than one year. While the structural changes of tumor cell surface carbohydrates are known to be associated with invasive behavior of tumor cells, the cell surface glycoproteins to differentiate the low‐ and high‐grade glioma cells can be potential diagnostic markers and therapeutic targets for GBMs. In the present study, lectin arrays consisting of eight lectins were employed to explore cell surface carbohydrate expression patterns on low‐grade oligodendroglioma cells (Hs683) and GBM cells (T98G). Griffonia simplicifolia I (GS I) was found to selectively bind to T98G cells and not to Hs683 cells. For identification of the glioblastoma‐specific cell surface markers, the glycoproteins from each cell type were captured by a GS I lectin column and analyzed by LC‐MS/MS. The identified proteins from the two cell types were quantified using label‐free quantitative analysis based on spectral counting. Of cell surface glycoproteins showing significant increases in T98G cells, five proteins were selected for verification of both protein and glycosylation level changes using Western blot and GS I lectin‐based immunosorbent assay.     

High‐Throughput Chiral LC–MS/MS Method Using Overlapping Injection Mode for the Determination of Pantoprazole Enantiomers in Human Plasma with Application to Pharmacokinetic Study

A sensitive and high‐throughput chiral liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of R‐pantoprazole and S‐pantoprazole in human plasma. Sample extraction was carried out by using ethyl acetate liquid–liquid extraction in 96‐well plate format. The separation of pantoprazole enantiomers was performed on a CHIRALCEL OJ‐RH column and an overlapping injection mode was used to achieve a run time of 5.0 min/sample. The mobile phase consisted of 1) 10 mM ammonium acetate in methanol: acetonitrile (1:1, v/v) and 2) 20 mM ammonium acetate in water. Isocratic elution was used with flow rate at 500 μL/min. The enantiomers were quantified on a triple‐quadrupole mass spectrometer under multiple reaction monitoring (MRM) mode with m/z 382.1/230.0 for pantoprazole and m/z 388.4/230.1 for pantoprazole‐d7. Linearity from 20.0 to 5000 ng/mL was established for each enantiomer (r² > 0.99). Extraction recovery ranged from 91.7% to 96.4% for R‐pantoprazole and from 92.5% to 96.5% for S‐pantoprazole and the IS‐normalized matrix factor was 0.98 to 1.07 for R‐pantoprazole and S‐pantoprazole, respectively. The method was demonstrated with acceptable accuracy, precision, selectivity, and stability and the method was applied to support a pharmacokinetic study of a phase I clinical trial of racemic pantoprazole in healthy Chinese subjects. Chirality 28:569–575, 2016. © 2016 Wiley Periodicals, Inc.     

Enantioselective Separation and Simultaneous Determination of Tolperisone and Eperisone in Rat Plasma by LC‐MS/MS

Tolperisone and eperisone used as muscle relaxants possess one chiral center each and exist as two optical isomers for each drug. Therefore, enantioselective assays to measure each enantiomer in biological matrices are of great importance. In the present study a simple and complete reverse‐phase liquid chromatography tandem mass spectrometric method for separation and enantioselective determination of tolperisone and eperisone in rat plasma was developed. The analytes were extracted from rat plasma by a simple protein precipitation method with acetonitrile as the extraction solvent. The enantioselective separation of analytes was achieved on a Cellulose Tris (4‐chloro‐3‐methylphenylcarbamate) chiral column with a mobile phase of acetonitrile: 10 mM ammonium acetate in an isocratic mode of elution and mass spectrometric detection. The calibration curve for each enantiomer was found to be linear over 0.2 to 20 ng/mL for each enantiomer. The proposed method exhibited good intra‐ and interday precision (% CV) ranged between 0.95–6.05% and 1.11–8.21%, respectively. The intra‐ and interday accuracy for the proposed assay method ranged between 94.0–100.5% and 92.7–102.1%, respectively. The proposed method was validated as per regulatory guidelines. Chirality 25:622–627, 2013. © 2013 Wiley Periodicals, Inc.     

Glycoproteins of the Chromaffin Granule Membrane: Separation by Two-Dimensional Electrophoresis and Identification by Lectin Binding

The proteins of highly purified chromaffin-granule membranes were separated by one- or two-dimensional electrophoresis, then transferred to nitrocellulose sheets; glycosylation was investigated by binding of several different radioiodinated lectins. Over 20 different glycosylated components were identified; comparison with mitochondrial and microsomal fractions suggested that most of the major glycoproteins are genuine components of the chromaffin granule membrane, rather than contaminants originating in other organelles. Two-dimensional electrophoresis revealed heterogeneity within several of the glycoproteins, and this is ascribed to differences in the state of glycosylation, on the basis of shifts in electrophoretic mobility produced by treatment with neuraminidase. Neuraminidase treatment of chromaffin granule membranes also enhances the binding of many lectins. The identities of the lectin-binding bands are discussed: neither cytochrome b561 nor the F1-like ATPase appears to be glycosylated. Chromogranin A, although a glycoprotein, does not bind any of the lectins tested, but a number of concanavalin-A binding proteins, as well as dopamine beta-hydroxylase, are present in the chromaffin granule lysate.     

Identification and Confirmation of Differentially Expressed Fucosylated Glycoproteins in the Serum of Ovarian Cancer Patients Using a Lectin Array and LC-MS/MS

In order to discover potential glycoprotein biomarkers in ovarian cancer, we applied a lectin array and Exactag labeling based quantitative glycoproteomics approach. A lectin array strategy was used to detect overall lectin-specific glycosylation changes in serum proteins from patients with ovarian cancer and those with benign conditions. Lectins, which showed significant differential response for fucosylation, were used to extract glycoproteins that had been labeled using isobaric chemical tags. The glycoproteins were then identified and quantified by LC-MS/MS, and five glycoproteins were found to be differentially expressed in the serum of ovarian cancer patients compared to benign diseases. The differentially expressed glycoproteins were further confirmed by lectin-ELISA and ELISA assay. Corticosteroid-binding globulin (CBG), serum amyloid p component (SAP), complement factor B (CFAB), and histidine-rich glycoprotein (HRG) were identified as potential markers for differentiating ovarian cancer from benign diseases or healthy controls. A combination of CBG and HRG (AUC = 0.825) showed comparable performance to CA125 (AUC = 0.829) in differentiating early stage ovarian cancer from healthy controls. The combination of CBG, SAP, and CA125 showed improved performance for distinguishing stage III ovarian cancer from benign diseases compared to CA125 alone. The ability of CBG, SAP, HRG, and CFAB to differentiate the serum of ovarian cancer patients from that of controls was tested using an independent set of samples. Our findings suggest that glycoprotein modifications may be a means to identify novel diagnostic markers for detection of ovarian cancer.     

Analysis of Oxcarbazepine and the 10‐Hydroxycarbazepine Enantiomers in Plasma by LC‐MS/MS: Application in a Pharmacokinetic Study

Oxcarbazepine is a second‐generation antiepileptic drug indicated as monotherapy or adjunctive therapy in the treatment of partial seizures or generalized tonic–clonic seizures in adults and children. It undergoes rapid presystemic reduction with formation of the active metabolite 10‐hydroxycarbazepine (MHD), which has a chiral center at position 10, with the enantiomers (S)‐(+)‐ and R‐(?)‐MHD showing similar antiepileptic effects. This study presents the development and validation of a method of sequential analysis of oxcarbazepine and MHD enantiomers in plasma using liquid chromatography with tandem mass spectrometry (LC‐MS/MS). Aliquots of 100 μL of plasma were extracted with a mixture of methyl tert‐butyl ether: dichloromethane (2:1). The separation of oxcarbazepine and the MHD enantiomers was obtained on a chiral phase Chiralcel OD‐H column, using a mixture of hexane:ethanol:isopropanol (80:15:5, v/v/v) as mobile phase at a flow rate of 1.3 mL/min with a split ratio of 1:5, and quantification was performed by LC‐MS/MS. The limit of quantification was 12.5 ng oxcarbazepine and 31.25 ng of each MHD enantiomer/mL of plasma. The method was applied in the study of kinetic disposition of oxcarbazepine and the MHD enantiomers in the steady state after oral administration of 300 mg/12 h oxcarbazepine in a healthy volunteer. The maximum plasma concentration of oxcarbazepine was 1.2 µg/mL at 0.75 h. The kinetic disposition of MHD is enantioselective, with a higher proportion of the S‐(+)‐MHD enantiomer compared to R‐(?)‐MHD and an AUC^0‐12 _{S‐(+)/R‐(?)} ratio of 5.44. Chirality 25:897–903, 2013. © 2013 Wiley Periodicals, Inc.     

Targeted On‐line SPE‐LC‐MS/MS Assay for the Quantitation of 12 Apolipoproteins from Human Blood

Laborious sample pretreatment of biological samples represents the most limiting factor for the translation of targeted proteomics assays from research to clinical routine. An optimized method for the simultaneous quantitation of 12 major apolipoproteins (apos) combining on‐line SPE and fast LC‐MS/MS analysis in 6.5 min total run time was developed, reducing the manual sample pretreatment time of 3 μL serum or plasma by 60%. Within‐run and between‐day imprecisions below 10 and 15% (n = 10) and high recovery rates (94–131%) were obtained applying the high‐throughput setup. High‐quality porcine trypsin was used, which outperformed cost‐effective bovine trypsin regarding digestion efficiency. Comparisons with immunoassays and another LC‐MS/MS assay demonstrated good correlation (Pearson's R: 0.81–0.98). Further, requirements on sample quality concerning sampling, processing, and long‐term storage up to 1 year were investigated revealing significant influences of the applied sampling material and coagulant on quantitation results. Apo profiles of 1339 subjects of the LIFE‐Adult‐Study were associated with lifestyle and physiological parameters as well as establish parameters of lipid metabolism (e.g., triglycerides, cholesterol). Besides gender effects, most significant impact was seen regarding lipid‐lowering medication. In conclusion, this novel highly standardized, high‐throughput targeted proteomics assay utilizes a fast, simultaneous analysis of 12 apos from least sample amounts.     

Stereoselective Determination of Metoprolol and its Metabolite α‐Hydroxymetoprolol in Plasma by LC‐MS/MS: Application to Pharmacokinetics during Pregnancy

Metoprolol is available for clinical use as a racemic mixture. The S‐(?)‐metoprolol enantiomer is the one expressing higher activity in the blockade of the β₁‐adrenergic receptor. The α‐hydroxymetoprolol metabolite also has activity in the blockade of the β₁‐adrenergic receptor. The present study describes the development and validation of a stereoselective method for sequential analysis of metoprolol and of α‐hydroxymetoprolol in plasma using high‐performance liquid chromatography with tandem mass spectrometry (LC‐MS/MS). 1‐ml aliquots of plasma were extracted with dichloromethane : diisopropyl ether (1:1, v/v). Metoprolol enantiomers and α‐hydroxymetoprolol isomers were separated on a Chiralpak AD column (Daicel Chemical Industries, New York, NY, USA) and quantitated by LC‐MS/MS. The limit of quantitation obtained was 0.2 ng of each metoprolol enantiomer/ml plasma and 0.1 ng/ml of each α‐hydroxymetoprolol isomer/ml plasma. The method was applied to the study of kinetic disposition of metoprolol in plasma samples collected up to 24 h after the administration of a single oral dose of 100‐mg metoprolol tartrate to a hypertensive parturient with a gestational age of 42 weeks. The clinical study showed that the metoprolol pharmakokinetics is enantioselective, with the observation of higher area under the curve (AUC)^0?∞ values for S‐(?)‐metoprolol (AUC_S‐(?)/AUC_R‐(+) = 1.81) and the favoring of the formation of the new chiral center 1′R of α‐hydroxymetoprolol (AUC^0?∞_1′R/1′S = 2.78). Chirality, 25:1–7, 2013. © 2012 Wiley Periodicals, Inc.     

Stereoselective analysis of labetalol in human plasma by LC‐MS/MS: Application to pharmacokinetics

Labetalol is clinically available as a mixture of two racemates (four stereoisomers). The stereoisomer (R,R) has as main activity the β₁‐antagonism and the stereoisomer (S,R) is highly selective for the α₁ adrenoceptor and is responsible for most of the α‐blocker activity. In the present investigation, a method for the analysis of labetalol stereoisomers in human plasma was developed and applied to pharmacokinetic studies. Plasma samples (0.5 ml) were extracted with methyl tert‐butyl ether at pH 9.5. The four labetalol stereoisomers were analyzed by LC‐MS/MS on a Chirobiotic® V column using a mobile phase consisting of methanol, acetic acid, and diethylamine, with a recovery of more than 90% for all four. The quantitation limit was 0.5 ng/ml and linearity was observed at 250 ng/ml plasma for each stereoisomer. Studies of precision and accuracy presented coefficients of variation and percentage inaccuracy of less than 15%, indicating that the method is precise and accurate. The method was applied to the study of the kinetic disposition of labetalol over a period of 12 h after oral administration of a single 100 mg dose to a hypertensive pregnant woman. The clinical study revealed stereoselectivity in the pharmacokinetics of labetalol, with a lower plasma proportion for the active stereoisomers (R,R)‐labetalol and (S,R)‐labetalol. The stereoselectivity observed after oral administration is due to the hepatic metabolism and the first pass effect, with an AUC_(R,R)/AUC_(S,S) ratio of 0.5. Chirality, 2009. © 2008 Wiley‐Liss, Inc.     

Multiplexed LC‐MS/MS analysis of horse plasma proteins to study doping in sport

The development of protein biomarkers for the indirect detection of doping in horse is a potential solution to doping threats such as gene and protein doping. A method for biomarker candidate discovery in horse plasma is presented using targeted analysis of proteotypic peptides from horse proteins. These peptides were first identified in a novel list of the abundant proteins in horse plasma. To monitor these peptides, an LC‐MS/MS method using multiple reaction monitoring was developed to study the quantity of 49 proteins in horse plasma in a single run. The method was optimised and validated, and then applied to a population of race‐horses to study protein variance within a population. The method was finally applied to longitudinal time courses of horse plasma collected after administration of an anabolic steroid to demonstrate utility for hypothesis‐driven discovery of doping biomarker candidates.     

Identification of Differentially Expressed Water‐insoluble Proteins in the Encystment Process of Colpoda cucullus by Two‐dimensional Electrophoresis and LC‐MS/MS Analysis

In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h–1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water‐insoluble proteins by two‐dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween‐80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60‐kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55‐kDa protein (p55; actin) and a 49‐kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50‐kDa protein (p50d; α‐tubulin), a 25‐kDa protein (p25; α‐tubulin) and a 52‐kDa protein (p52c; β‐tubulin) was enhanced.     

Identification of common and differential mechanisms of glomerulus and tubule senescence in 24‐month‐old rats by quantitative LC–MS/MS

Kidney aging together with related renal disease had become a major clinical problem. Understanding the mechanisms of aging was important for suspending senescence and decreasing the incidence of aging‐related diseases. In the present work, 24‐month‐old F344 rats were used as aging rats and 3‐month‐old rats were used as young controls. Senescence‐associated‐β‐galactosidase staining results showed that the degree of senescence in renal tubules was more severe than that in glomeruli. We performed quantitative LC–MS to assess the differential protein expression profiles of senescent glomeruli and tubules. Bioinformatics analysis showed that aging, response to oxidative stress, nucleotide metabolism, amine acid metabolism, and inflammatory response were common mechanisms of glomerulus and tubule senescence. Differentially expressed proteins network mediated Golgi vesicle transport, actin filament based process, and regulation of cell death were associated with tubule senescence. More importantly, we found that the changes of four and a half LIM protein 2 (FHL2) were opposite in senescent glomeruli and tubules, and FHL2 could regulate p16 by suppressing T‐box 3, which was involved in regulation of senescence in glomeruli and tubules. In conclusion, we assessed the mechanisms of senescence in aging glomeruli and tubules, and the results yielded new insight into kidney senescence.     

HTII-280, a Biomarker Specific to the Apical Plasma Membrane of Human Lung Alveolar Type II Cells

The pulmonary alveolar epithelium is composed of two morphologically distinct cell types, type I (TI) and type II (TII) cells. Alveolar TII cells synthesize, secrete, and recycle surfactant components; contain ion transporters; and secrete immune effector molecules. In response to alveolar injury, TII cells have the capacity to act as progenitor cells, proliferating and transdifferentiating into TI cells. Although various proteins are associated with TII cells, a plasma membrane marker specific to human TII cells that would be useful for identification in tissue and for isolating this cell type has not been described previously. We devised a strategy to produce a monoclonal antibody (MAb) specific to the apical surface of human TII cells and developed an MAb that appears to be specific for human TII cells. The antibody recognizes a 280- to 300-kDa protein, HTII-280, which has the biochemical characteristics of an integral membrane protein. HTII-280 is detected by week 11 of gestation and is developmentally regulated. HTII-280 is useful for isolating human TII cells with purities and viabilities >95%. HTII-280 is likely to be a useful morphological and biochemical marker of human TII cells that may help to advance our understanding of various lung pathological conditions, including the origin and development of various lung tumors. (J Histochem Cytochem 58:891–901, 2010)