Shotgun proteomic methods involving isobaric tagging of peptides enable high-throughput proteomic analysis. iTRAQ reagents allow simultaneous identification and quantitation of proteins in four different samples using tandem mass spectrometry (MS). In this article, we provide a brief description of proteome analysis using iTRAQ reagents and review the current applications of these reagents in p...

Boron (B) phytotoxicity affects cereal-growing regions worldwide. Although B-tolerant barley (Hordeum vulgare) germplasm is available, molecules responsible for this tolerance mechanism have not been defined. We describe and use a new comparative proteomic technique, iTRAQ peptide tagging (iTRAQ), to compare the abundances of proteins from B-tolerant and -intolerant barley plants from a 'Clippe...

We assess the reliability of isobaric-tags for relative and absolute quantitation (iTRAQ), based on different types of replicate analyses taking into account technical, experimental, and biological variations. In total, 10 iTRAQ experiments were analyzed across three domains of life involving Saccharomyces cerevisiae KAY446, Sulfolobus solfataricus P2, and Synechocystis sp. PCC 6803. The covera...

High resolution proteomics approaches have been successfully utilized for the comprehensive characterization of the cell proteome. However, in the case of quantitative proteomics an open question still remains, which quantification strategy is best suited for identification of biologically relevant changes, especially in clinical specimens. In this study, a thorough comparison of a label-free a...

There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO-S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRA...

While many data are available on genes encoding proteins for degradation of hydrocarbons in bacteria, the impact of alkane on transporter protein expression is unclear. Pseudomonas aeruginosa SJTD-1 is a strain that can consume medium- and long-chain n-alkanes. In order to study the proteins involved in n-octadecane uptake, we use iTRAQ and label free comparative proteomics analysis to identify...

The ability to distinguish between phosphopeptides of high and low stoichiometry is essential to discover the true extent of protein phosphorylation. We here extend the strategy whereby a peptide sample is briefly split in two identical parts and differentially labeled preceding the phosphatase treatment of one part. Our use of isobaric tags for relative and absolute quantitation (iTRAQ) marks ...

Ceramides play a crucial role in divergent signaling events, including differentiation, senescence, proliferation, and apoptosis. Ceramides are a minor lipid component in terms of content; thus, highly sensitive detection is required for accurate quantification. The recently developed isobaric tags for relative and absolute quantitation (iTRAQ) method enables a precise comparison of both protei...

Iron (Fe) is required by plants for basic redox reactions in photosynthesis and respiration, and for many other key enzymatic reactions in biological processes. Fe homeostatic mechanisms have evolved in plants to enable the uptake and sequestration of Fe in cells. To elucidate the network of proteins that regulate Fe homeostasis and transport, we optimized the iTRAQ-OFFGEL method to identify an...