Shotgun proteomics using the iTRAQ isobaric tags

Abstract

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 proteomic studies. We also compare different aspects of protein identification including protein sequence coverage and proteome coverage obtained using iTRAQ reagents with those using other shotgun proteomic techniques. We briefly discuss the issue of isotope purity correction in measured peak areas during protein quantitation using iTRAQ reagents. Finally, we conclude with some of the current challenges in MS-based proteomic analysis that are limiting protein identifications obtained by different shotgun proteomic methods.

Tissue Proteomics for the Next Decade? Towards a Molecular Dimension in Histology

“…LC-MS based quantitative methods such as label free quantification (Higgs et al, 2005;Neilson et al, 2011;Wang et al, 2006), spectral counting quantification (Neilson et al, 2011), intensity based quantification (Bondarenko et al, 2002;Christin et al, 2011), and also labeling techniques were developed for off-gel analyses. Labeling methods such as isobaric tag for relative and absolute quantification (iTRAQ) (Aggarwal et al, 2006;Chen et al, 2010;DeSouza et al, 2005;Garbis et al, 2008;Muraoka et al, 2012;Wang et al, 2012), and the recently developed isotope coded protein labeling coupled to immunoprecipitation (ICPL-IP) (Vogt et al, 2013), both relying on the labeling of the primary amino groups and lysine residues, were developed. Isotope coded affinity tag (ICAT) (DeSouza et al, 2005), that labels cysteine residues was also applied for tissue proteomics.…”

Proteomics in Diagnosis: Past, Present and Future

“…iTRAQ is another technique to perform absolute or relative quantification of protein in gel free system. This technology uses an NHS ester derivative to modify primary amino groups by linking a mass balance group (carbonyl group) and a reporter group (based on N-methylpiperazine) to proteolytic peptides via the formation of an amide bond [44]. Multidimensional Protein Identification Technology (MudPIT) is yet another emerging proteomics technique.…”

Using Laboratory Information Management Systems as central part of a proteomics data workflow

“…The project team particularly announces support of peptide quantization experiments (e.g. iTRAQ [31,34] and SILAC [35][36][37]). Furthermore, the installation routine is now considerably simplified.…”

“…Several quantification possibilities are already available for ProSE including analysis of iTRAQ [31,34] and DIGE [50] surveys. Label-free quantification can be performed on the basis of the msInspect output.…”