Proteomic analysis of transducin β-subunit structural heterogeneity

James W. Clack, Martha Juhl, Carol A. Rice, Junyu Li, Frank Witzmann

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Partially purified transducin was resolved using two-dimensional gel electrophoresis (2-DE). Peptide mass fingerprinting of several different spots believed to correspond to the 37 kDa β-subunit of transducin (Tβ) was performed. Spots were excised and proteolyzed using modified trypsin. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was performed on the peptide mixture resulting from each spot. As many as six spots with different p/, ranging from 5.2 to 6.1, were observed when separated using 2-DE. MALDI peptide mass fingerprinting determined with high probability that all of the spots were the same gene product, guanine nucleotide-binding protein GI/GS/GT β-subunit 1 (GNB1; Tβ1). This suggested that post-translational modification was responsible for the differences in p/. Phosphorylation experiments showed that at least one Tβ1 spot was phosphorylated in vitro with [γ-32P]ATP by an endogenous kinase. Treatment of Tβ with alkaline phosphatase caused a large change in the spot pattern of Tβ, suggesting that phosphorylated Tβ is a substrate for alkaline phosphatase. We conclude that Tβ1 constitutes over 99% of the Tβexpressed in bovine rod outer segments and displays structural heterogeneity that is due to post-translational modification. We also conclude that some, but not all, of the heterogeneity observed is due to phosphorylation of Tb1.

Original languageEnglish
Pages (from-to)3493-3499
Number of pages7
JournalElectrophoresis
Volume24
Issue number19-20
StatePublished - Oct 2003

Fingerprint

Transducin
Peptide Mapping
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Post Translational Protein Processing
Proteomics
Alkaline Phosphatase
Phosphorylation
Rod Cell Outer Segment
Peptides
Guanine Nucleotides
Electrophoresis, Gel, Two-Dimensional
Trypsin
Carrier Proteins
Phosphotransferases
Adenosine Triphosphate
Electrophoresis
Ionization
Mass spectrometry
Desorption
Genes

Keywords

  • G-Proteins
  • Photoreceptors
  • Post-translational modification
  • Proteomics
  • Signal transduction

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Clack, J. W., Juhl, M., Rice, C. A., Li, J., & Witzmann, F. (2003). Proteomic analysis of transducin β-subunit structural heterogeneity. Electrophoresis, 24(19-20), 3493-3499.

Proteomic analysis of transducin β-subunit structural heterogeneity. / Clack, James W.; Juhl, Martha; Rice, Carol A.; Li, Junyu; Witzmann, Frank.

In: Electrophoresis, Vol. 24, No. 19-20, 10.2003, p. 3493-3499.

Research output: Contribution to journalArticle

Clack, JW, Juhl, M, Rice, CA, Li, J & Witzmann, F 2003, 'Proteomic analysis of transducin β-subunit structural heterogeneity', Electrophoresis, vol. 24, no. 19-20, pp. 3493-3499.
Clack JW, Juhl M, Rice CA, Li J, Witzmann F. Proteomic analysis of transducin β-subunit structural heterogeneity. Electrophoresis. 2003 Oct;24(19-20):3493-3499.
Clack, James W. ; Juhl, Martha ; Rice, Carol A. ; Li, Junyu ; Witzmann, Frank. / Proteomic analysis of transducin β-subunit structural heterogeneity. In: Electrophoresis. 2003 ; Vol. 24, No. 19-20. pp. 3493-3499.
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