Proteome analysis of a human uveal melanoma primary cell culture by 2-DE and MS

We present here the first proteomics analysis of uveal melanoma (UM) cells. These cells represent a good model for the identification of polypeptide markers, which could be developed as diagnostic tools. UM is the most common primary intraocular tumour in adults. In contrast to other cancers, the survival rate of patients with this malignancy has changed little over the past few decades; a better understanding of the molecular biology of UM oncogenesis and metastasis is needed to build the basis for the identification of novel drug targets. In the study presented here, proteins from a UM primary cell culture were separated by 2-DE using a pI 3-10 gradient; 270 spots were analysed by LC-MS/MS, identifying 683 proteins derived from 393 different genes. Of those, 69 (18%) are related to cancer processes involving cell division, proliferation, invasion, metastasis, oncogenesis, drug resistance and others. To our knowledge, 96% of the proteins identified, including 16 hypothetical proteins, have never been reported in UM before. This study represents the first step towards the establishment of a UM protein database as a valuable resource for the study of this malignancy.     

Glycan profiling of monoclonal antibodies using zwitterionic-type hydrophilic interaction chromatography coupled with electrospray ionization mass spectrometry detection

We present a new method for the analysis of glycans enzymatically released from monoclonal antibodies (MAbs) employing a zwitterionic-type hydrophilic interaction chromatography (ZIC–HILIC) column coupled with electrospray ionization mass spectrometry (ESI–MS). Both native and reduced glycans were analyzed, and the developed procedure was compared with a standard HILIC procedure used in the pharmaceutical industry whereby fluorescent-labeled glycans are analyzed using a TSK Amide-80 column coupled with fluorescence detection. The separation of isobaric alditol oligosaccharides present in monoclonal antibodies and ribonuclease B is demonstrated, and ZIC–HILIC is shown to have good capability for structural recognition. Glycan profiles obtained with the ZIC–HILIC column and ESI–MS provided detailed information on MAb glycosylation, including identification of some less abundant glycan species, and are consistent with the profiles generated with the standard procedure. This new ZIC–HILIC method offers a simpler and faster approach for glycosylation analysis of therapeutic antibodies.     

Quantification of CLR1401, a novel alkylphosphocholine anticancer agent,in rat plasma by hydrophilic interaction liquid chromatography–tandem mass spectrometric detection

A rapid and specific LC–MS/MS based bioanalytical method was developed and validated for the determination of 18-(p-iodophenyl)octadecyl phosphocholine (CLR1401), a novel phosphocholine drug candidate, in rat plasma. The optimal chromatographic behavior of CLR1401 was achieved on a Kromasil silica column (50 mm × 3 mm, 5 μm) under hydrophilic interaction chromatography. The total LC analysis time per injection was 2.8 min with a flow rate of 1.5 mL/min under gradient elution. Liquid–liquid extraction in a 96-well format using ethyl acetate was developed and applied for method validation and sample analysis. The method validation was conducted over the curve range of 2.00–1000 ng/mL using 0.0500 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤ 5.9% relative standard deviation (RSD) and −10.8 to −1.4% relative error (RE). The method was successfully applied to determine the toxicokinetics of CLR1401 in rats from three dose groups of 0.4, 4.0, and 10.0 mg/kg/day via intravenous administration.     

Flavonoids from the leaves of Cyclanthera pedata: two new malonyl derivatives

Reversed-phase HPLC coupled with electrospray MS has been used for the simultaneous separation and determination of flavonoid metabolites in leaves of Cyclanthera pedata, an edible Peruvian plant mainly used in South America for its anti-inflammatory, hypoglycaemic and hypocholesterolaemic properties. The flavonoid content of the leaves of C. pedata was compared qualitatively and quantitatively with that of the fruits. The isolation and structural characterisation by MS and NMR of two new minor components of the fruits, namely, 6-C-fucopyranosyl-(3-malonyl)-chrysin and 6-C-fucopyranosyl-(4-malonyl)-chrysin, are described.     

Rapid identification of vinca alkaloids by direct-injection electrospray ionisation tandem mass spectrometry and confirmation by high-performance liquid chromatography-mass spectrometry

A simple and rapid method for the identification of Vinca alkaloids from a crude extract of Catharanthus roseus G. Don (Apocynaceae) by direct-injection electrospray ionisation (ESI) and tandem mass spectrometry (MS/MS) has been developed. The alkaloids vindoline, vindolidine, vincristine and vinblastine were evaluated in a commercial extract of C. roseus using this method. Catharanthine and its isomers 19S-vindolinine and vindolinine were detected in the commercial product by direct injection ESI/MS/MS and confirmed by preparation and by HPLC-ESI/MS. For the characterisation of different fragment fingerprints, ESI/MS/MS is a sensitive, rapid and convenient technique by which to identify some constituents in complex and mixed plant extracts.