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- W3109602287 abstract "593 Tubulin, the constitutive protein of microtubules, is an α/β heterodimer. In humans, there are six α-tubulin isotypes and seven β-tubulin isotypes, each the product of a unique gene. Heterogenous distribution of the isotypes has been observed among different tissues. The functional significance and the factors regulating this differential expression of tubulin isotypes are poorly understood. In cancer cells, alterations in tubulin isotype composition and tubulin mutations have been associated with resistance to drugs that interact with microtubules. Mass spectrometry-based methods have been developed in our laboratory to analyze tubulin isotype expression, mutations and posttranslational modifications in human cell lines and tissues. Since mass spectrometry is an inherently non-quantitative technique, we have used stable isotope-containing amino acids in cell culture (SILAC) to differentially label proteins in Taxol-sensitive- and resistant cell lines . In the SILAC method, two cell lines are cultured for several generations in media containing either deuterated leucine (d3-Leu) or normal leucine (d0-Leu). After incorporation, equal amounts of total cytosolic proteins from each cell line were mixed and tubulin was isolated by Taxol-induced polymerization. For relative quantitation of total α- and β-tubulin levels, the region containing tubulin was excised from a SDS-PAGE gel and subjected to trypsin digestion followed by MALDI-TOF mass spectrometry. Depending on the number of leucine residues contained in a tryptic peptide, this peptide was represented by two m/z peaks separated by a multiple of 3 Da in the mass spectra. The signal intensity of each peak in these doublets was used to calculate the relative abundance of a given isotype within the two cell lines. We compared the tubulin content of MDA-MB-231 cell line and MDA-MB-231.K20T, a Taxol-resistant cell line bearing a Glu198Gly mutation in βI-tubulin. Our data indicated that both α- and β-tubulin expression increased about 1.5-fold in MDA-MB-231.K20T. For relative quantitation of individual tubulin isotype levels, tubulin isotypes were separated by IEF and each gel band containing a particular isotype was analyzed by tryptic mass mapping. In MDA-MB-231.K20T, βIII-tubulin was increased by 2-fold, βIVb decreased by 0.7-fold, Kα1 increased by 2-fold and α6 increased by 1.3-fold. In the case of βI, a small amount of wild type protein was detected in MDA-MB-231.K20T (2.5-fold less than in MDA-MB-231), whereas most of this isotype was represented by the more basic mutated protein. We are utilizing the SILAC approach to analyze tubulin isotype expression in other cell lines, and also the differential expression of proteins associated with the microtubule cytoskeleton. This quantitative proteomic study will highlight those changes in cellular microtubule composition that may be related to Taxol resistance." @default.
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- W3109602287 date "2004-04-01" @default.
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- W3109602287 title "Quantitative proteomics of α-and β-tubulin isotype expression in human cancer cells" @default.
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