Analysis of complex protein samples by two-dimensional electrophoresis (2-DE) is often more difficult in the presence of a few predominant proteins. In plasma, proteins such as albumin mask proteins of lower abundance, as well as significantly limiting the amount of protein that can be loaded onto the immobilized pH gradient strip. In this paper the Gradiflow, a preparative electrophoresis system, has been used to deplete human plasma of the highly abundant protein albumin under native and denatured conditions. A three step protocol incorporating a charge separation to collect proteins with an isoelectric point greater than albumin and two size separations to isolate proteins larger and smaller than albumin, was used. When the albumin depleted fractions were analysed on pH 3-10 2-DE gels, proteins that were masked by albumin were revealed and proteins not seen in the unfractionated plasma sample were visualised. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry analysis confirmed the identification of the protein that lies beneath albumin to be C4B-binding protein alpha chain. The liquid fractions from the Gradiflow separations were also analysed by liquid chromatography-tandem mass spectrometry to confirm the proteins were separated according to their size and charge mobility in an electric field.

One of the major challenges facing protein analysis is the dynamic range of protein expression within massively complex samples (Corthals, G. L. et al.., Electrophoresis 2000, 21, 1104-1115). In plasma this difference is as great as ten orders of magnitude, and this is currently beyond the range of detection achievable by any of the analytical techniques. Plasma has the additional challenge of having a few highly abundant proteins, such as albumin, which mask the detection of lower abundance and biologically significant proteins. The use of the Gradiflow BF400 as a fractionation tool to deplete highly abundant albumin from human plasma is reported here. A sequential three-step protocol was performed on five plasma samples as part of the International Plasma Proteome Project organised by the HUPO; four containing different anticoagulants: EDTA, citrate, heparin and a control sample (NIBSC); and a serum sample. Plasma from an alternate source also underwent fractionation and served as an in-house control. Time modulation between 1 and 7 h was observed for the depletion of albumin from these samples. Following albumin depletion, each fraction was trypsin-digested and the peptides were fractionated further using a 2-D LC-MS/MS. Differences in the total number of proteins identified for each sample were also noted.

Small cell lung cancer (SCLC) responds to treatment with cisplatin and etoposide, but relapse is rapid and survival rates are low. Our aims were to determine the mechanisms of resistance and the potential for paclitaxel (Taxol) to overcome any drug or radiation resistance. To mimic clinical treatment, H69 SCLC cells, representative of the classic form of the disease, and H82 cells, with the phenotype of the more resistant variant disease, were treated intermittently with 100 ng/ml cisplatin or 500 ng/ml etoposide (approximate IC50 drug doses) to produce stable sublines. Drug and radiation resistance were determined using the MTT assay. Protein expression was determined by Western blot. The effect of paclitaxel on drug resistance was determined by cytotoxicity assays. Intermittent 4-day treatment with 100 ng/ml cisplatin caused 2- to 3-fold resistance to cisplatin (n=5; p<0.05), and 2- to 5-fold cross resistance to etoposide, alkylating drugs, the Vinca drugs and radiation. Resistance was mediated primarily by changes in glutathione metabolism and was not associated with changes in MRP2 transport protein. Treatment with etoposide (500 ng/ml) produced cells with 2-fold resistance to etoposide (n=5; p<0.05). Cross-resistance was limited and mediated by decreased topoisomerase IIalpha. Treatment of both drug-resistant sublines with a maximal non-cytotoxic dose of paclitaxel sensitized them to other drugs and to radiation, although this treatment had no effect on the parental H69 or H82 cells. We conclude that paclitaxel may play an important role in the treatment of refractory SCLC.

The Gradiflow trade mark, a preparative electrophoresis instrument capable of separating proteins on the basis of their size or charge, was used to separate whole cell lysates, prepared from bakers yeast (Saccharomyces cerevisiae) and Chinese snow pea seeds (Pisum sativum macrocarpon), into protein fractions of different pH regions. Both broad and narrow range (with a difference of approximately 1 pH unit) pH fractions were obtained. Analysis of the protein fractions by isoelectric focusing gels and two-dimensional (2-D) polyacrylamide gel electrophoresis indicated minimal overlap between the pH fractions. Further, when the prefractionated acidic samples were analyzed on pH 4-7 immobilized pH gradient 2-D gels, improved resolution of the proteins within the chosen pH region was achieved compared to the unfractionated samples. This study demonstrates that the Gradiflow could be used as a preparative electrophoresis tool for the isolation of proteins into distinct pH fractions.

Summary. Relative to the commonly used anthracyclines, little is known about idarubicin and the development of multidrug resistance. We have previously shown the K562/ IDA subline resulting from intermittent treatment of the K562 human leukaemia cell line with 20 ng/ml idarubicin did not develop multidrug resistance but became more sensitive to etoposide. Additional similar treatments of this subline produced the K562/IDA20 subline which partially retained its etoposide sensitivity although these cells expressed P-glycoprotein and were resistant to paclitaxel. Sensitization to etoposide was associated with increased decatenation activity of topoisomerase II, although there were no changes in topoisomerase IIa expression or formation of etoposide-dependent cleavable complexes. In comparison, the K562/IDA10 subline produced by intermittent treatment of the K562 cells, ®rstly with 5 ng/ml then 10 ng/ml idarubicin, showed no detectable expression of P-glycoprotein, decreased topoisomerase IIa expression and increased resistance to etoposide and amsacrine, but not to idarubicin or genistein. Even though intermittent treatment with idarubicin caused increased drug resistance in both sublines, they remained sensitive to idarubicin. Therefore the potential of idarubicin as a substitute for other anthracyclines in the treatment of cancer warrants further investigation.

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Sensitization of drug-resistant cells by paclitaxel was not associated with its ability to cause a G(2)/M block in the cell cycle. Sensitization by paclitaxel and vinblastine, but not navelbine, which preferentially targets mitotic tubulin, suggests that sensitization may involve changes in the tubulin-dependent intracellular transport processes rather than changes in mitotic tubulin and the G(2)/M block.

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