Separation of human erythrocyte membrane associated proteins with one-dimensional and two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry

Low Teck Yew, Teck Keong Seow, Maxey C.M. Chung

Research output: Contribution to journalArticle

107 Citations (Scopus)

Abstract

A classical proteomic analysis was used to establish a reference map of proteins associated with healthy human erythrocyte ghosts. Following osmotic lysis and differential centrifugation, ghost proteins were separated by either one-dimensional gel electrophoresis (1-DE) or two-dimensional gel electrophoresis (2-DE). Selected protein bands or spots were excised and trypsinized before mass spectrometric analyses and data mining was performed using the SWISS-PROT and NCBI nonredundant databases. A total of 102 protein spots from a 2-D gel were successfully identified. These corresponded to 59 distinct polypeptides with the remaining 43 being isoforms. As for the 1-D gel, 44 polypeptides were identified, of which 19 were also found on the 2-D gel. Most of the 19 common polypeptides were membrane cytoskeletal proteins that are often referred to as the "band" proteins. The remaining 25 polypeptides that were found exclusively on 1-D gels were proteins with high hydrophobicity (e.g., sorbitol dehydrogenase and glucose transporter) and high molecular mass (e.g., Kell blood group glycoprotein and Janus-kinase 2). A higher number of signaling proteins was also identified on 1-D gels compared to 2-D gels. These included Ras, cAMP dependent protein kinase and TGF-beta receptor type 1 precursor.

Original languageEnglish
Pages (from-to)1229-1239
Number of pages11
JournalProteomics
Volume2
Issue number9
DOIs
Publication statusPublished - 1 Sep 2002
Externally publishedYes

Fingerprint

Electrophoresis, Gel, Two-Dimensional
Erythrocyte Membrane
Electrophoresis
Ionization
Mass spectrometry
Desorption
Mass Spectrometry
Membrane Proteins
Lasers
Gels
Proteins
Peptides
L-Iditol 2-Dehydrogenase
Janus Kinase 2
Transforming Growth Factor beta1
Transforming Growth Factor beta Receptors
Protein Databases
Cytoskeletal Proteins
Data Mining
Facilitative Glucose Transport Proteins

Keywords

  • Human erythrocyte membrane protein
  • Matrix-assisted laser desorption ionization-time of flight mass spectrometry
  • One-dimensional gel electrophoresis
  • Two-dimensional gel electrophoresis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

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title = "Separation of human erythrocyte membrane associated proteins with one-dimensional and two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry",
abstract = "A classical proteomic analysis was used to establish a reference map of proteins associated with healthy human erythrocyte ghosts. Following osmotic lysis and differential centrifugation, ghost proteins were separated by either one-dimensional gel electrophoresis (1-DE) or two-dimensional gel electrophoresis (2-DE). Selected protein bands or spots were excised and trypsinized before mass spectrometric analyses and data mining was performed using the SWISS-PROT and NCBI nonredundant databases. A total of 102 protein spots from a 2-D gel were successfully identified. These corresponded to 59 distinct polypeptides with the remaining 43 being isoforms. As for the 1-D gel, 44 polypeptides were identified, of which 19 were also found on the 2-D gel. Most of the 19 common polypeptides were membrane cytoskeletal proteins that are often referred to as the {"}band{"} proteins. The remaining 25 polypeptides that were found exclusively on 1-D gels were proteins with high hydrophobicity (e.g., sorbitol dehydrogenase and glucose transporter) and high molecular mass (e.g., Kell blood group glycoprotein and Janus-kinase 2). A higher number of signaling proteins was also identified on 1-D gels compared to 2-D gels. These included Ras, cAMP dependent protein kinase and TGF-beta receptor type 1 precursor.",
keywords = "Human erythrocyte membrane protein, Matrix-assisted laser desorption ionization-time of flight mass spectrometry, One-dimensional gel electrophoresis, Two-dimensional gel electrophoresis",
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