Journal of Chromatography B (v.840, #1)

Changes of the cortex proteome and Apolipoprotein E in transgenic mouse models of Alzheimer's Disease by O. Carrette; J.A. Burgess; P.R. Burkhard; C. Lang; M. Côte; N. Rodrigo; D.F. Hochstrasser; J.-C. Sanchez (1-9).
Transgenic mice carrying human Amyloid Precursor Protein mutations present amyloid plaque deposition in the brain upon aging. In this study, we characterized the changes of cortex proteome and endogenous Apolipoprotein E in these mice. Differential analysis of two-dimensional electrophoresis images revealed spots altered upon aging, transgene addition and plaque deposition. Alpha-synuclein and cytochrome oxidase polypeptide Va were up-regulated in transgenic mice. Upon aging, expression of ATP synthase α, α enolase, UMP-CMP kinase, and dihydropyrimidinase like-2 protein was modified. These proteins and their modification probably play a role in the amyloid aggregate formation in these mice.
Keywords: Two-dimensional electrophoresis; Alzheimer's disease; Transgenic mice; Cortex;

Differentiation of HELLP patients from healthy pregnant women by proteome analysis—On the way towards a clinical marker set by J.C. Heitner; C. Koy; M. Kreutzer; B. Gerber; T. Reimer; M.O. Glocker (10-19).
As the pathogenesis of the HELLP-syndrome is unknown, a complex consideration regarding the changes in the plasma as the main transport medium in the body is of great benefit because it is well available and can rapidly be investigated in the clinics. Besides that, the liver which is early affected in HELLP-syndrome produces the main part of plasma proteins. For the purpose of our study plasma protein abundances from patients with HELLP-syndrome and from control individuals were determined before and after delivery. In the differential analysis using two-dimensional gel electrophoresis, six areas with variable protein spot intensities were detected. The reference gel that we developed for HELLP plasma samples integrates the changes of plasma proteins when comparing HELLP patients to healthy women prior to and after delivery. A specific plasma protein profile for the HELLP-syndrome was generated involving protein areas that contain inter-alpha-trypsin inhibitor heavy chain H4, kininogen 1, fibrinogen gamma chain, transthyretin, haptoglobins, and serum amyloid A with statistically significant expression differences when compared to controls. The most striking difference between the majority of the gels from HELLP patients and the gels from non-HELLP samples were clearly overexpressed protein spots at about 11 kDa which were identified as serum amyloid A (SAA). This differential expression was validated and quantitatively assayed by ELISA measurements against human SAA in plasma. Our results show that significant differences in SAA expressions between healthy controls and HELLP patients were obtained, that could function as markers for the HELLP-syndrome. According to our data it is possible to draw a line of separation with no overlap between the HELLP group for which SAA plasma levels were found to be above 3.51 mg/L and the non-HELLP groups in which SAA plasma levels were below 3.51 mg/L. It now is possible to clinically elucidate if the differentially expressed proteins are suited for longitudinal studies concerning both, to function as markers or perhaps even as disease predictors that might become relevant for diagnostic tests.
Keywords: HELLP-syndrome; Proteomics; 2-DE; Two-dimensional gel electrophorsesis; Mass spectrometry; SAA ELISA;

Peptidomics and proteomics studies of transformed lymphocytes from patients mutated for the eukaryotic initiation factor 2B by Anne Fogli; Claire Malinverni; Lynne Thadikkaran; Patricia Combes; Frédéric Perret; David Crettaz; Jean Daniel Tissot; Odile Boespflug-Tanguy; Reto Stöcklin; Philippe Bulet (20-28).
Mutations in the ubiquitous factor eIF2B involved in protein synthesis and its regulation have been reported in human brain genetic disorders. In order to analyse the functional consequences of the mutations and to find specific biomarkers of eIF2B-related disorders, proteomics and peptidomics studies were performed on lymphoblasts from eIF2B-mutated patients versus healthy patients. Curiously, following two-dimensional gel electrophoresis and mass fingerprints, mutations in the eIF2B complex did not significantly affect the proteome of the mutated lymphoblasts extracts. However, liquid chromatography based peptidomics studies revealed one apparently instable candidate compound in five out of the six mutated lymphoblastoid cell lines investigated.
Keywords: Lymphoblasts; eIF2B-related disorders; CACH/VWM leukodystrophy; eIF2B-pathies; Peptidomics; Proteomics; Differential analyses; Mass spectrometry;

Concanavalin A, boronic acid and Wheat germ agglutinin functionalized magnetic micro-particles were developed to enrich glycosylated peptides and proteins. The bead functionalities were validated according to their specificity by analyses of model proteins. Validated beads were employed for the enrichment of glycosylated human serum proteins. Eluted glycoproteins were digested by trypsin and the resulting peptides were purified by magnetic MB-HIC C8 beads. Each fraction was analyzed by MALDI-TOF MS and single peaks were subjected to MALDI-TOF/TOF MS with the objective to identify the respective proteins by database search. Search results revealed overlapping profiles of known serum glycoproteins.
Keywords: Serum glycoproteins; MALDI-TOF/TOF MS; Magnetic particles;

2-O-α-d-glucopyranosyl-6-O-hexadecanoyl-l-ascorbic acid (6-sPalm-AA-2G), a novel stable lipophilic ascorbic acid derivative, was hydrolyzed to 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G), ascorbyl 6-palmitate (6-sPalm-AA) and ascorbic acid (AA) with α-glucosidase and lipase. An HPLC method for the simultaneous determination of AA, AA-2G, 6-sPalm-AA and 6-sPalm-AA-2G was developed using a cyanopropyl column with an isocratic solution of methanol–phosphate buffer (pH 2.1) (65:35, v/v) containing 20 mg/l of dithiothreitol at a detection wavelength of 240 nm. The calibration curves were found to be linear in the range of 10–200 μM. Linear regression analysis of the data demonstrated the efficacy of the method in terms of precision and accuracy. This method was satisfactorily applied to the determination of 6-sPalm-AA-2G and its three metabolites in a 6-sPalm-AA-2G solution treated with purified enzymes or a small intestine post-mitochondrial supernatant and to the separation of novel stable lipophilic AA derivatives other than 6-sPalm-AA-2G and their metabolites. AA, AA-2G and other well-known stable AA derivatives, ascorbic acid 2-phosphate and ascorbic acid 2-sulfate, were also separated under the same conditions. The results show that the procedure is rapid and simple and that it can be employed for in vitro metabolic analysis of various AA derivatives.
Keywords: Novel stable lipophilic ascorbic acid derivative (6-acyl-AA-2G); AA-2G; Ascorbic acid; Ascorbyl 6-palmitate; Cyano column; HPLC;

A reverse-phase HPLC method based on ion-pair formation with UV detection was set up for the simultaneous determination of gemcitabine diphosphate (dFdCDP) and triphosphate (dFdCTP) in human cells. The separation was achieved on a Tracer Excel ODSA column (100 mm × 4.6 mm i.d., 3 μm particle size) at room temperature. Nine nucleotides were separated by isocratic elution in 26 min. Accuracy, linearity, sensitivity and precision studies for dFdCDP, dFdCTP, adenosine diphosphate (ADP) and triphosphate (ATP) validated this method. This assay was used to provide data from gemcitabine treated patients and in vitro grown human cancer cells.
Keywords: dFdCTP; dFdCDP; Reverse-phase HPLC; Peripheral blood mononuclear cells; Gemcitabine;

A reversed-phase HPLC method for the simultaneous quantitative determination of mandelic acid enantiomers (MA) and phenylglyoxylic acid (PGA) in urine is described. MA and PGA were extracted with ethyl acetate from urine at acidic pH and derivatized with S-(−)-1-(1-naphthyl) ethylamine. A ZORBAX SB-C18 column (250 mm × 4.6 mm i.d., 5 μm, Agilent, USA) was used with a mobile phase composed of methanol–10 mmol/L phosphate buffer [pH 2.5 (65:35, v/v)] at a flow-rate of 0.8 ml/min. Detection was set at UV wavelength of 254 nm. The mean absolute recoveries were 94.2%, 91.9%, 92.5% and 86.3% for S-MA, R-MA, PGA and salicylic acid (I.S.), respectively. The intra- and inter-day precisions determined at three different concentrations ranged from 2.8% to 4.8%, 0.7% to 7.7% and 1.3% to 6.8%, respectively. The lower limits of detection for MA enantiomers and PGA in urine were 1 μg/ml and the lower limits of quantification were 5 μg/ml (R.S.D. < 10%, n  = 5). The method has been applied to determine the urinary excretion of MA enantiomers and PGA from Sprague–Dawley rats after orally administered with styrene.
Keywords: Mandelic acid enantiomers; Phenylglyoxylic acid; Styrene; Indirect enantioseparation; Precolumn derivatization;

LC–MS/MS assay and dog pharmacokinetics of the dimeric pyrrolobenzodiazepine SJG-136 (NSC 694501) by Sarah A. Buhrow; Joel M. Reid; Lee Jia; Renee M. McGovern; Joseph M. Covey; Dean J. Kobs; Irma M. Grossi; Matthew M. Ames (56-62).
The dimeric pyrrolobenzodiazepine SJG-136 (NSC 694501) has potent in vitro cytotoxicity and in vivo antitumor activity. SJG-136 binds in the minor groove of DNA and produces G–G interstrand cross-links via reactive N10–C11/N10′–Cll′ imine/carbinolamine moieties. We have developed a sensitive, specific liquid chromatography tandem mass spectrometry (LC/MS/MS) method for the quantitative determination of SJG-136 in plasma. SJG-136 was isolated by solid phase extraction through a C8 column, reverse-phase HPLC separation was accomplished on a C18 column with isocratic elution and MS/MS detection, monitoring the m/z 557–m/z 476 transition after electrospray ionization. The linear range and lower limit of quantitation from plasma standard curves were 2.8–1800 nM, and 5 nM, respectively. SJG-136 plasma protein binding was species-dependent. Values of the unbound fraction in human, rat and mouse were 25%, 16.2% and <1%, respectively. Protein binding was saturable in dog plasma where the unbound fraction increased from 10.8% to 22.3% over a 22–720 nM concentration range. SJG-136 pharmacokinetics after a single intravenous dose were best fit to a two-compartment open model with elimination half-life and plasma clearance values of 97 min and 6.1 mL/min/kg, respectively. SJG-136 did not accumulate in plasma following intravenous administration of 1.0 μg/kg doses for five consecutive days.
Keywords: LC/MS/MS; Pharmacokinetics; Anticancer agents; Benzodiazepine;

Immobilized hemin affinity chromatography as a probe for proteins having potentiality to bind with heme by Renqiang Li; Fengyi Jiang; Xiaofen Zhang; Yao Chen; Ling Fang (63-68).
After Sepharose 4B polymer beads were activated by using epichlorohydrin, hemin was binded with them to prepare an immobilized hemin affinity chromatography column. The coupling rate of this column was very high, more than 0.25 mg hemin could be fixed by 1 g of wet Sepharose 4B beads. The column equilibrated with deionized water and eluated with pH 3.0 NaAc–HAc buffer was applied to capture the proteins in human serum, earthworm body and Bacillus subtilis cells. Three polypeptides in human serum were captured, one of which was verified as serum albumin after comparison to the control. At least one polypeptide in earthworm body, two in Bacillus subtilis cells displayed the powerful binding specificity to hemin. Our experiments demonstrated that the immobilized hemin affinity chromatography was available as a probe for some proteins having potentiality to bind with heme.
Keywords: Hemin; Affinity chromatography; Sepharose 4B; Human serum; Earthworm; Bacillus subtilis;

17α-Hydroxyprogesterone is a metabolic precursor of cortisol; elevated levels of 17α-hydroxyprogesterone are indicative of congenital adrenal hyperplasia. Traditional determination by immunoassay is plagued by poor antibody specificity, resulting in significant interferences. This study explores an LC–MS/MS method for the quantitation of 17OHP in serum. Deuterated 17α-hydroxyprogesterone was added as internal standard, followed by solid-phase extraction, HPLC separation with a C16-amide reverse-phase column with run time of 7 min, and quantification by MS/MS (positive electrospray ionisation) in the selected reaction monitoring mode (SRM). Transitions monitored were 331 > 109 for the analyte and 339 > 113 for the deuterated internal standard. Intra-assay precision (%R.S.D.) was 7.4% at 7 nmol/L, inter-assay precision (%R.S.D.) at 2, 7 and 27 nmol/L was 15.4, 10.0 and 7.9% and accuracy at 0.9 nmol/L was 100%. The method was linear from 0.156 to 80 nmol/L. Lower limit of quantitation was 0.2 nmol/L, providing meaningful data for patients within normal range as well as those with elevated levels.
Keywords: LC–MS/MS; CAH; Congenital adrenal hyperplasia; 17-Hydroxy progesterone; RIA; SPE;