Separation and identification of platinum adducts with DNA nucleotides by capillary zone electrophoresis and capillary zone electrophoresis coupled to mass spectrometry.

Platinum adducts are supposed to be the cytotoxic lesions in DNA after platinum-containing anticancer therapy. Various adducts are formed upon interaction of platinum complexes with nucleotides, but contribution of individual adducts to antitumor activity and toxicity of platinum complexes still remains to be examined. A capillary zone electrophoresis (CZE) method is described that is suitable to separate individual platinum adducts. We investigated the formation of adducts following the reaction of cis-diamminedichloroplatinum (II) (cisplatin) with various DNA nucleotides. Baseline separation of unmodified and modified nucleotides (adducts) was achieved using uncoated fused-silica capillaries and basic separation buffers. In order to elucidate the observed peak pattern, a coupled CZE-electrospray ionization-mass spectrometry (ESI)-MS approach was applied. After incubation of mononucleotides with cisplatin, monochloro, monoaqua and bifunctional adduct species were detected. Consequently, the migration order of nucleotides and individual platinum adducts could be determined. Moreover, the time-dependent conversion from monochloro to monoaqua and subsequently to bifunctional adducts was monitored. In conclusion, individual platinum adducts were separated by CZE and identified by CZE-ESI-MS. Formation and conversion of distinct species were confirmed. Potential applications comprise studies of novel platinum complexes, investigations of platinum-adduct formation with DNA, and determination of platinum-DNA adducts in cells.

Chip-based capillary electrophoresis with an electrodeless nanospray interface.

A sheathless and electrodeless nanospray interface has been used to interface a polycarbonate capillary electrophoresis (CE) chip to a mass spectrometer (MS). The chip was made of two flat polycarbonate plates which were bolted together. Channels were imprinted in one of the plates with metal wires, using a hydraulic press. A short tapered capillary connected to the chip was used as the nanospray emitter. The advantage of this electrodeless interface is that it was not necessary to apply a electrospray voltage to the chip or the nanospray emitter. Instead, the CE voltage already applied to the buffer compartment on the chip, to drive the electrophoresis, was used to generate the spray also. A low conductivity buffer of 1.25 mmol/L ammonium acetate in 80% methanol was used to obtain a large electric field across the buffer channel. The performance of the device was evaluated by analyzing a mixture of three beta-agonists Relative standard deviation (RSD) values obtained were between 4.8 and 5.0%. A sample concentration of 40 nmol/L resulted in a signal-to-noise ratio of 2 to 5 for the different components. Compared to a conventional CE analysis in a fused silica capillary with UV detection, only a minor loss of resolution was observed, which can be attributed to the design of the chip.

Characterization of recombinant cytokine fragments using isotachophoresis-capillary zone electrophoresis, reversed-phase high performance liquid chromatography, and mass spectrometry.

PURPOSE: Capillary zone electrophoresis with isotachophoretic sample preconcentration (ITP-CZE) and reversed-phase high performance liquid chromatography (RP-HPLC) with UV detection and on-line coupling to electrospray-ionization mass spectrometry were investigated for their potential to separate and identify fragments of recombinant human interleukin-6 formed during acidic stress of the parent protein. RESULTS: Based on the orthogonal separation principles governing ITP-CZE and RP-HPLC, different peak patterns were observed using both methods. The selectivity of ESI-MS allowed identification of several co-migrating compounds. Data obtained by on-line ESI-MS were compared to results from off-line investigations by MALDI-TOF-MS performed with single fractions collected from the RP-HPLC system. Cleavage of the protein backbone occurred preferably at acid-labile Asp-sites. The total amount of rhIL-6 needed for ITP-CZE-ESI-MS identification of all fragments was only in the upper femtomole range, while RP-HPLC required amounts of protein three orders of magnitude higher. On the other hand, the low CE sample volume opposes the collection of fractions to perform off-line analysis. CONCLUSIONS: Growing acceptance of CE with on-line MS detection for pharrmaceutical quality control of proteins is expected.

Utility of isotachophoresis-capillary zone electrophoresis, mass spectrometry and high-performance size-exclusion chromatography for monitoring of interleukin-6 dimer formation.

The utility of isotachophoresis-capillary zone electrophoresis (ITP-CZE) and high-performance size-exclusion chromatography (HPSEC) was investigated for determination of dimeric and monomeric recombinant human interleukin-6 (rhIL-6). Using ITP-CZE heterogeneity of dimeric rhIL-6 could be revealed resolving two peaks in the electropherograms, while with HPSEC dimeric rhIL-6 eluted as one homogeneous fraction. Both protein forms were monitored during incubation of monomeric rhIL-6 at different pH and temperature. The selectivity of counterflow ITP-CZE in conjunction with the low concentration determination limits enabled reanalysis of HPSEC fractions for identification of the dimer in the electropherograms. Both ITP-CZE and HPSEC were shown to be suitable to monitor the dimerization of rhIL-6, similar monomer-to-dimer peak area ratios were obtained throughout the incubation. Dimer formation kinetics increased with decreasing pH and with increasing temperature, it was entirely suppressed at neutral pH and room temperature. In contrast to HPSEC, ITP-CZE enabled separation of further still unidentified artifacts apparently formed during incubation of rhIL-6. CZE analysis in conjunction with electrospray ionization mass spectrometry revealed the non-covalent binding character of the dimeric rhIL-6 complex and facilitated interpretation of the electropherograms.

Monitoring of chemotherapy-induced proteinuria using capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) was investigated for its suitability to monitor proteinuria occurring during nephrotoxic drug therapy. Urine samples of tumor patients receiving chemotherapy consisting of carboplatin, etoposide, and ifosfamide were concentrated and desalted in microconcentrators and analyzed in two different alkaline CZE buffer systems. Reduction of electroosmotic flow (EOF) by the addition of putrescine increased the number of resolved protein peaks. Both CZE methods were linear between 2.5 and 50 microg/ml, exhibited satisfactory precision (relative standard deviation <10%) and were suitable for monitor the time course of proteinuria after chemotherapy administration. In contrast to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), CZE detected interindividual differences in protein patterns. Whereas these differences hampered a direct quantification of proteins in urine, they may contain information on the type or extent of kidney damage.