Analysis of tubulin isotypes and mutations from taxol-resistant cells by combined isoelectrofocusing and mass spectrometry

Pascal Verdier-Pinard, Fang Wang, Laura Martello, Berta Burd, George A. Orr, Susan Band Horwitz

Research output: Contribution to journalArticle

89 Citations (Scopus)

Abstract

Six human α-tubulin and seven human β-tubulin isotypes, each of which can undergo posttranslational modifications, have been detected by the reverse transcriptase - polymerase chain reaction. This repertoire of tubulin isotypes plays a role in development and in the building of specialized microtubule-based structures. In cell lines, the relationship between resistance to microtubule-interacting drugs and altered tubulin isotype expression profiles is often established by quantitation of cDNA and/or Western blot analysis. Tubulin mutations in major isotypes are detected by sequencing cDNA, but more analysis of expression of tubulin mutations at the protein level, to assess their role in drug resistance, is needed. We utilized a Taxol-based purification and high-resolution isoelectrofocusing combined with a mass spectrometry-based analysis of tubulin. This approach has allowed the separation and relative quantitation of tubulin isotypes having a difference in isoelectric point values of 0.01, without the need for two-dimensional gel electrophoresis. The specificity of tubulin isotype antibodies also has been established. In cell lines resistant to microtubule-stabilizing drugs that express heterozygous tubulin mutations, the relative amount of mutant tubulin expression has been determined. In these cell lines, the absence of βII- and βIVa-tubulin has been demonstrated, and an increased level of expression of βIII-tubulin in resistant cells has been confirmed, indicating that this tubulin isotype is a unique marker of resistance.

Original languageEnglish (US)
Pages (from-to)5349-5357
Number of pages9
JournalBiochemistry
Volume42
Issue number18
DOIs
StatePublished - May 13 2003

Fingerprint

Tubulin
Paclitaxel
Mass spectrometry
Mass Spectrometry
Mutation
Microtubules
Cells
Cell Line
Complementary DNA
Pharmaceutical Preparations
Polymerase chain reaction
RNA-Directed DNA Polymerase
Electrophoresis, Gel, Two-Dimensional
Isoelectric Point
Post Translational Protein Processing
Electrophoresis
Reverse Transcriptase Polymerase Chain Reaction
Drug Resistance
Purification
Western Blotting

ASJC Scopus subject areas

  • Biochemistry

Cite this

Analysis of tubulin isotypes and mutations from taxol-resistant cells by combined isoelectrofocusing and mass spectrometry. / Verdier-Pinard, Pascal; Wang, Fang; Martello, Laura; Burd, Berta; Orr, George A.; Band Horwitz, Susan.

In: Biochemistry, Vol. 42, No. 18, 13.05.2003, p. 5349-5357.

Research output: Contribution to journalArticle

Verdier-Pinard, Pascal ; Wang, Fang ; Martello, Laura ; Burd, Berta ; Orr, George A. ; Band Horwitz, Susan. / Analysis of tubulin isotypes and mutations from taxol-resistant cells by combined isoelectrofocusing and mass spectrometry. In: Biochemistry. 2003 ; Vol. 42, No. 18. pp. 5349-5357.
@article{7cc3bd036a5a49d3b3ae3b13fcf30d02,
title = "Analysis of tubulin isotypes and mutations from taxol-resistant cells by combined isoelectrofocusing and mass spectrometry",
abstract = "Six human α-tubulin and seven human β-tubulin isotypes, each of which can undergo posttranslational modifications, have been detected by the reverse transcriptase - polymerase chain reaction. This repertoire of tubulin isotypes plays a role in development and in the building of specialized microtubule-based structures. In cell lines, the relationship between resistance to microtubule-interacting drugs and altered tubulin isotype expression profiles is often established by quantitation of cDNA and/or Western blot analysis. Tubulin mutations in major isotypes are detected by sequencing cDNA, but more analysis of expression of tubulin mutations at the protein level, to assess their role in drug resistance, is needed. We utilized a Taxol-based purification and high-resolution isoelectrofocusing combined with a mass spectrometry-based analysis of tubulin. This approach has allowed the separation and relative quantitation of tubulin isotypes having a difference in isoelectric point values of 0.01, without the need for two-dimensional gel electrophoresis. The specificity of tubulin isotype antibodies also has been established. In cell lines resistant to microtubule-stabilizing drugs that express heterozygous tubulin mutations, the relative amount of mutant tubulin expression has been determined. In these cell lines, the absence of βII- and βIVa-tubulin has been demonstrated, and an increased level of expression of βIII-tubulin in resistant cells has been confirmed, indicating that this tubulin isotype is a unique marker of resistance.",
author = "Pascal Verdier-Pinard and Fang Wang and Laura Martello and Berta Burd and Orr, {George A.} and {Band Horwitz}, Susan",
year = "2003",
month = "5",
day = "13",
doi = "10.1021/bi027293o",
language = "English (US)",
volume = "42",
pages = "5349--5357",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "18",

}

TY - JOUR

T1 - Analysis of tubulin isotypes and mutations from taxol-resistant cells by combined isoelectrofocusing and mass spectrometry

AU - Verdier-Pinard, Pascal

AU - Wang, Fang

AU - Martello, Laura

AU - Burd, Berta

AU - Orr, George A.

AU - Band Horwitz, Susan

PY - 2003/5/13

Y1 - 2003/5/13

N2 - Six human α-tubulin and seven human β-tubulin isotypes, each of which can undergo posttranslational modifications, have been detected by the reverse transcriptase - polymerase chain reaction. This repertoire of tubulin isotypes plays a role in development and in the building of specialized microtubule-based structures. In cell lines, the relationship between resistance to microtubule-interacting drugs and altered tubulin isotype expression profiles is often established by quantitation of cDNA and/or Western blot analysis. Tubulin mutations in major isotypes are detected by sequencing cDNA, but more analysis of expression of tubulin mutations at the protein level, to assess their role in drug resistance, is needed. We utilized a Taxol-based purification and high-resolution isoelectrofocusing combined with a mass spectrometry-based analysis of tubulin. This approach has allowed the separation and relative quantitation of tubulin isotypes having a difference in isoelectric point values of 0.01, without the need for two-dimensional gel electrophoresis. The specificity of tubulin isotype antibodies also has been established. In cell lines resistant to microtubule-stabilizing drugs that express heterozygous tubulin mutations, the relative amount of mutant tubulin expression has been determined. In these cell lines, the absence of βII- and βIVa-tubulin has been demonstrated, and an increased level of expression of βIII-tubulin in resistant cells has been confirmed, indicating that this tubulin isotype is a unique marker of resistance.

AB - Six human α-tubulin and seven human β-tubulin isotypes, each of which can undergo posttranslational modifications, have been detected by the reverse transcriptase - polymerase chain reaction. This repertoire of tubulin isotypes plays a role in development and in the building of specialized microtubule-based structures. In cell lines, the relationship between resistance to microtubule-interacting drugs and altered tubulin isotype expression profiles is often established by quantitation of cDNA and/or Western blot analysis. Tubulin mutations in major isotypes are detected by sequencing cDNA, but more analysis of expression of tubulin mutations at the protein level, to assess their role in drug resistance, is needed. We utilized a Taxol-based purification and high-resolution isoelectrofocusing combined with a mass spectrometry-based analysis of tubulin. This approach has allowed the separation and relative quantitation of tubulin isotypes having a difference in isoelectric point values of 0.01, without the need for two-dimensional gel electrophoresis. The specificity of tubulin isotype antibodies also has been established. In cell lines resistant to microtubule-stabilizing drugs that express heterozygous tubulin mutations, the relative amount of mutant tubulin expression has been determined. In these cell lines, the absence of βII- and βIVa-tubulin has been demonstrated, and an increased level of expression of βIII-tubulin in resistant cells has been confirmed, indicating that this tubulin isotype is a unique marker of resistance.

UR - http://www.scopus.com/inward/record.url?scp=0038219598&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0038219598&partnerID=8YFLogxK

U2 - 10.1021/bi027293o

DO - 10.1021/bi027293o

M3 - Article

C2 - 12731876

AN - SCOPUS:0038219598

VL - 42

SP - 5349

EP - 5357

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 18

ER -