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. 2006 Sep 1;11(3):326–337. doi: 10.2478/s11658-006-0026-2

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

Jose Chou 1,3,4, Pankaj K Choudhary 2,3,4, Steven R Goodman 1,3,4,5,✉
PMCID: PMC6472844  PMID: 16847560

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.

Key words: Proteomics, Cleavable ICAT, Ion trap mass spectrometry, RBC membrane skeleton, Sickle cell

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Abbreviations used

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

Footnotes

Invited paper

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