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Protein profiling of sickle cell versus control RBC core membrane skeletons by ICAT technology and tandem mass spectrometry

Cellular & Molecular Biology Letters volume 11, pages 326–337 (2006)Cite this article

Abstract

A proteomic approach using a cleavable ICAT reagent and nano-LC ESI tandem mass spectrometry was used to perform protein profiling of core RBC membrane skeleton proteins between sickle cell patients (SS) and controls (AA), and determine the efficacy of this technology. The data was validated through Peptide/Protein Prophet and protein ratios were calculated through ASAPratio. Through an ANOVA test, it was determined that there is no significant difference in the mean ratios from control populations (AA1/AA2) and sickle cell versus control populations (AA/SS). The mean ratios were not significantly different from 1.0 in either comparison for the core skeleton proteins (α spectrin, β spectrin, band 4.1 and actin). On the natural-log scale, the variation (standard deviation) of the method was determined to be 14.1% and the variation contributed by the samples was 13.8% which together give a total variation of 19.7% in the ratios.

Abbreviations

ASAPratio:

automated statistical analysis of protein abundance ratios+

cICAT:

cleavable isotope coded affinity tag

CID:

collision induced dissociation

2D DIGE:

two dimension differential gel electrophoresis

ESI:

electrospray ionization source

ICAT:

isotope coded affinity tag

LC:

liquid chromatography

RBC:

red blood cell

SD:

standard deviation

SILAC:

stable isotope labeled amino acids in cell culture

WBCs:

white blood cells

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Affiliations

  1. Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas, USA

    Jose Chou & Steven R. Goodman

  2. Department of Mathematical Sciences, University of Texas at Dallas, Richardson, Texas, USA

    Pankaj K. Choudhary

  3. The Institute of Biomedical Sciences and Technology, University of Texas at Dallas, Richardson, Texas, USA

    Jose Chou, Pankaj K. Choudhary & Steven R. Goodman

  4. University of Texas at Dallas, Richardson, Texas, USA

    Jose Chou, Pankaj K. Choudhary & Steven R. Goodman

  5. Department of Cell Biology, UT Southwestern Medical Center, Dallas, Texas, USA

    Steven R. Goodman

Authors
  1. Jose Chou
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  2. Pankaj K. Choudhary
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  3. Steven R. Goodman
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Correspondence to Steven R. Goodman.

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Chou, J., Choudhary, P.K. & Goodman, S.R. Protein profiling of sickle cell versus control RBC core membrane skeletons by ICAT technology and tandem mass spectrometry. Cell Mol Biol Lett 11, 326–337 (2006). https://doi.org/10.2478/s11658-006-0026-2

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Key words

  • Proteomics
  • Cleavable ICAT
  • Ion trap mass spectrometry
  • RBC membrane skeleton
  • Sickle cell

Cellular & Molecular Biology Letters

ISSN: 1689-1392

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