Shotgun proteomics using the iTRAQ isobaric tags.

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.

[1]  T. Gingeras,et al.  Prokaryotic RNA preparation methods useful for high density array analysis: comparison of two approaches. , 2001, Nucleic acids research.

[2]  Andrew Emili,et al.  De novo peptide sequencing and quantitative profiling of complex protein mixtures using mass-coded abundance tagging , 2002, Nature Biotechnology.

[3]  Steven C Hall,et al.  Assessing the effects of diurnal variation on the composition of human parotid saliva: quantitative analysis of native peptides using iTRAQ reagents. , 2005, Analytical chemistry.

[4]  Steven P Gygi,et al.  Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations , 2005, Nature Methods.

[5]  B. Chait,et al.  Preferential Fragmentation of Protonated Gas-Phase Peptide Ions Adjacent to Acidic Amino Acid Residues , 1995 .

[6]  T. Colgan,et al.  Search for cancer markers from endometrial tissues using differentially labeled tags iTRAQ and cICAT with multidimensional liquid chromatography and tandem mass spectrometry. , 2005, Journal of proteome research.

[7]  J. Yates,et al.  Large-scale analysis of the yeast proteome by multidimensional protein identification technology , 2001, Nature Biotechnology.

[8]  E. Wang,et al.  Simultaneous proteomic profiling of four different growth states of human fibroblasts, using amine-reactive isobaric tagging reagents and tandem mass spectrometry , 2006, Mechanisms of Ageing and Development.

[9]  C. Bessant,et al.  i-Tracker: For quantitative proteomics using iTRAQ™ , 2005, BMC Genomics.

[10]  X. Yao,et al.  Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus. , 2001, Analytical chemistry.

[11]  R. Becklin,et al.  Development of an LC-MALDI method for the analysis of protein complexes , 2004, Journal of the American Society for Mass Spectrometry.

[12]  Kelvin H Lee,et al.  A comparison of the consistency of proteome quantitation using two‐dimensional electrophoresis and shotgun isobaric tagging in Escherichia coli cells , 2005, Electrophoresis.

[13]  Kelvin H. Lee,et al.  Quantitative analysis of protein expression using amine‐specific isobaric tags in Escherichia coli cells expressing rhsA elements , 2005, Proteomics.

[14]  K. Parker,et al.  Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents*S , 2004, Molecular & Cellular Proteomics.

[15]  S. Gygi,et al.  Quantitative analysis of complex protein mixtures using isotope-coded affinity tags , 1999, Nature Biotechnology.

[16]  D. Lauffenburger,et al.  Time-resolved Mass Spectrometry of Tyrosine Phosphorylation Sites in the Epidermal Growth Factor Receptor Signaling Network Reveals Dynamic Modules*S , 2005, Molecular & Cellular Proteomics.

[17]  Rafael A. Irizarry,et al.  A Model-Based Background Adjustment for Oligonucleotide Expression Arrays , 2004 .

[18]  T. Veenstra,et al.  Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling. , 2001, Analytical chemistry.

[19]  Andrew H. Thompson,et al.  Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. , 2003, Analytical chemistry.