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J Biomol Tech. 2007 February; 18(1): 74.
PMCID: PMC2291909

P214-S Identification and Quantitation of Isobarically Labeled Proteins: A Comparison of Separation Strategies

Abstract

Current protocols for protein quantitation using iTRAQ reagents utilize a tryptic peptide–labeling strategy. While there are many advantages to this workflow, it precludes the effective use of protein-level separation techniques. We compare two protein-level labeling workflows. The first is in-gel digestion after SDS-PAGE, and the second is solution-phase digestion after size-exclusion chromatography. Chromatography offers increased sample load relative to electrophoresis and thus potentially increased access to proteins of lower abundance.

The study was conducted on a human cell line (U937) infected with dengue virus, a mosquito-borne flavivirus of the Flaviviridae family. Intact protein samples were reduced, alkylated, and labeled at the lysine residues with one of the isobaric iTRAQ reagent tags, pooled, and then separated by size-exclusion chromatography. After separation, the fractions were digested with trypsin. These workflows resulted in larger peptides and reduced complexity, as the proteins are digested only at the arginine residues. The digests were then analyzed by LC-MALDI on a 4800 MALDI TOF/TOF Analyzer. Initial data show approximately 750 proteins identified using the gel-based workflow, with a similar number of identifications from the size-exclusion data. The analysis will be focused on global protein expression changes as a result of infection in order to elicit information on how the virus competes with normal cellular mRNAs for the translational machinery of the cell.


Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities