Analytical characteristics of cleavable isotope-coded affinity tag-LC-tandem mass spectrometry for quantitative proteomic studies.

Abstract

Quantitative proteomic studies using cleavable isotope-coded affinity tags (cICAT) in concert with tandem mass spectrometry (MS/MS) permit unbiased comparisons between biologically distinct samples. We sought to determine the analytical characteristics of cICAT-based studies by examining the cumulative results of multiple, separate cICAT-based experiments involving human lymphoma-derived cells. We found that the number of identified proteins increased with larger numbers of fractions analyzed. The majority of proteins were identified by single peptides. Only 24 to 41% of the peptides contained cysteine residues, but 85% of the cysteine-containing peptides yielded quantification data. Approximately 28% of all identified proteins yielded quantification data, with 57% of these being differentially expressed by at least 1.5-fold. The quantification ratios of peptides for proteins with multiple quantified peptides were concordant in trend in 87% of instances. cICAT-labeled peptides identified proteins in all subcellular compartments without significant bias. Analysis of the flow-through fraction did not increase the number of peptides identified per protein. Our studies indicate that cICAT-LC-MS/MS yields quantifications primarily based on single peptides, and analysis of flow-through peptides does not contribute signifi-cantly to the results. Nevertheless, identifications based on single cICAT-labeled peptides with tryptic ends provide sufficiently reliable protein identifications and quantification information in cICAT-LC-MS/MS-based proteomic studies.

Cite this paper

@article{Vaughn2006AnalyticalCO, title={Analytical characteristics of cleavable isotope-coded affinity tag-LC-tandem mass spectrometry for quantitative proteomic studies.}, author={Cecily Vaughn and David K. Crockett and Megan Su Cheng Lim and Kojo S. J. Elenitoba-Johnson}, journal={The Journal of molecular diagnostics : JMD}, year={2006}, volume={8 4}, pages={513-20} }