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Comparative Evaluation of Two Isobaric Labeling Tags, Diart and Itraq † ‡ § † ‡ ‡ ‡ ⊥ ¶ ⊥ ¶ Zhen Chen, , , Quanhui Wang, , Liang Lin, Qi Tang, James L

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Comparative Evaluation of Two Isobaric Labeling Tags, Diart and Itraq † ‡ § † ‡ ‡ ‡ ⊥ ¶ ⊥ ¶ Zhen Chen, , , Quanhui Wang, , Liang Lin, Qi Tang, James L Article pubs.acs.org/ac Comparative Evaluation of Two Isobaric Labeling Tags, DiART and iTRAQ † ‡ § † ‡ ‡ ‡ ⊥ ¶ ⊥ ¶ Zhen Chen, , , Quanhui Wang, , Liang Lin, Qi Tang, James L. Edwards, , Shuwei Li,*, , † ‡ and Siqi Liu*, , † Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 101318, China ‡ BGI-Shenzhen, Shenzhen, 518083, China § Graduate University of the Chinese Academy of Sciences, Beijing, 100049, China ⊥ Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland 20850, United States ¶ Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, United States ABSTRACT: Isobaric tags have broad applications in both basic and translational research, as demonstrated by the widely used isobaric tag for relative and absolute quantitation (iTRAQ). Recent results from large-scale quantitative proteomics projects, however, indicate that protein quantifi- cation by iTRAQ is often biased in complex biological samples. Here, we report the application of another isobaric tag, deuterium isobaric amine reactive tag (DiART), for quantify- ing the proteome of Thermoanaerobacter tengcongensis (T. tengcongensis), a thermophilic bacterium first discovered in China. We compared the performance of DiART with iTRAQ from several different aspects, including their fragmentation mechanisms, the number of identified proteins, and the accuracy of quantification. Our results revealed that, as compared with iTRAQ, DiART yielded significantly stronger reporter ions, which did not reduce the number of identifiable peptides, but improved the signal-to- noise ratio (S/N) for quantification. Remarkably, we found that, under identical chromatography and mass spectrometry (MS) conditions, DiART exhibited less reporter ions ratio compression than iTRAQ, probably due to more reporter ions with higher intensities produced by DiART labeling. Taken together, we demonstrate that DiART is a valuable alternative of iTRAQ with enhanced performance for quantitative proteomics. ells are miniature biological factories where proteins and unpredictable factors in quantitative proteomics thus are C other biomolecules coordinate together to manipulate removed, and more accurate quantitative information could biological functions and maintain cellular homeostasis. This be achieved. These methods, on the basis of how proteins or exquisite balance can often be perturbed by abnormal peptides are labeled and quantified, can be grouped into two expression or altered activity of some key proteins in response large categories, mass-shift tags and isobaric tags. A peptide to certain biological stimuli. Exploring these key proteins is of labeled with mass-shift tags shows distinct peaks with a fixed great interest for biologists because the information can pave a mass difference in an MS spectrum, whose intensity reflects the way to the deep understanding of molecular mechanisms abundance of this peptide in original samples. Some popular underlying various biological processes. However, as there are techniques using mass-shift tags, such as stable isotope labeling thousands of proteins with numerous posttranslational with amino acids in cell culture (SILAC)2 and isotope-coded modifications and broad dynamic ranges in cells or tissues, it affinity tags (ICAT),3 have been generally employed in is not an easy job to find these responsive proteins with critical quantitative proteomics. On the other hand, approaches with functions. To address this challenge, approaches capable of isobaric tags have emerged in recently years and played a more detecting proteins in a high-throughput manner and at important role to quantify proteomes. Isobaric tags do not quantitative level are badly needed. introduce mass difference into labeled peptides because they Mass spectrometry (MS) is one of few methods with such are isotope-coded molecules with the same chemical structure capability and has therefore become a widely utilized tool for 4 1 and molecular weight. Their structure consists of a reporter profiling proteomes. Unfortunately, MS itself is not a good and a balancer containing different isotopes positioned in a way tool for quantitative measurement because its signal intensity is that the different masses of the reporters in a set of reagents are prone to the influence of many parameters, such as physical− compensated by those of the balancers. In addition, the chemical properties of analytes and matrix effects. To overcome this problem, stable isotope labeling approaches have been Received: December 26, 2011 developed to allow multiple biological samples to be analyzed Accepted: February 29, 2012 under the same chromatography and MS conditions. The Published: February 29, 2012 © 2012 American Chemical Society 2908 dx.doi.org/10.1021/ac203467q | Anal. Chem. 2012, 84, 2908−2915 Analytical Chemistry Article chemical bond linking the reporter and the balancer is ■ MATERIALS AND METHODS breakable under collision induced dissociation (CID) con- Materials. All solvents (HPLC-grade) were purchased from ditions in MS. Taken together, a peptide differentially labeled J. T. Baker (Philipsburg, NJ, USA). Sequence-grade trypsin was by a set of isobar tags, albeit displaying a single peak on an MS obtained from Promega (Madison, WI, USA). The chemicals spectrum, can yield a series of low-mass reporter ions for for culture media were from Oxoid (Basingstroke, Hampshire, quantification upon tandem mass spectrometry (MS/MS) UK). The DiART reagents were synthesized and prepared as fragmentation. Currently, there are two popular isobaric previously reported.11 The iTRAQ reagents Multiplex kit was reagents available in market, tandem mass tag (TMT)5 and 6 purchased from AB Sciex (Foster City, CA, USA). Other isobaric tag for relative and absolute quantitation (iTRAQ). chemicals were from Sigma-Aldrich (St. Louis, MO, USA). Up to eight samples can be quantified concurrently by these T. tengcongensis Growth. T. tengcongensis strain MB4T was reagents, thereby improving both the throughput and cultured in the media as previously described under four reproducibility of MS analysis. different temperatures, 55, 65, 75, and 80 °C, respectively.12 Most isotope labeling reagents for large-scale proteomics The bacterial cells were harvested at the middle stage of log 2 experiments do not use deuterium ( H) as one of the heavy phase when OD600 approximately reached 0.6. The cell pellets isotopes because 2H can introduce a detrimental chromato- were collected after centrifugation at 4000g at 4 °C, washed graphic shift in reverse phase liquid chromatograph (RP-LC) twice with an isotonic buffer (50 mM Tris-HCl, pH = 7.8), and and compromise the accuracy of quantification.7 Therefore, stored at −80 °C until use. TMT and iTRAQ tags only contain 13C, 15N, and 18O as coding Protein Extraction and Digestion. The cell pellets were isotopes, partly contributing to the high cost associated with suspended in the lysis buffer (8 M urea, 4% 3-[(3- their applications. Interestingly, the 2H-associated isotope effect cholamidopropyl)dimethylammonio]-1-propanesulfonate in RP-LC has been recently found to be negligible when 2H (CHAPS), 10 mM dithiothreitol (DTT), and 40 mM Tris− atoms are placed close to hydrophilic groups like tertiary HCl, pH = 8.0) and sonicated in ice. The proteins were amines.8 On the basis of this observation, we developed a set of reduced with 10 mM DTT at 56 °C and then alkylated by 55 isobaric reagents, deuterium isobaric amine reactive tag mM iodoacetamide in the dark at room temperature. The (DiART), and showed that they can quantify both proteins9 treated proteins were reprecipitated in 80% acetone at −20 °C and small molecule metabolites.10 In contrast to TMT and overnight and redissolved in 0.8 M urea and 500 mM iTRAQ, the synthesis of DiART is much easier, making it more tetraethylammonium bicarbonate (TEAB), pH 8.5. After cost-effective for applications that require labeling a large centrifuging at 12 000g at 4 °C, an aliquot of the supernatant quantity of samples.11 In addition, DiART reagents show high was taken for determination of protein concentration by the chemical and isotope purity, also thanks to their short synthetic Bradford method. The proteins in TEAB were digested by ° routes. trypsin at 37 C, and the solvent was removed by Speedvac. We previously tested the performance of DiART reagents on Isobaric Tag Labeling. Basically, the 6-plex DiART labeling was similar as previously reported with slight a simplified system: tryptic peptides digested from a mixture of 9 9 modifications. The DiART reagents (0.8 mg) were dissolved three proteins. To make DiART useful for the proteomic μ analysis of biological samples as well as for the proteomics in 48 L of anhydrous acetonitrile. The labeling reaction mixture contained 45 μL of the DiART reagent and 20 μLof community, here, we demonstrate the application of DiART on μ evaluating temperature-dependent protein expression in the peptide solution (100 g of peptide in 500 mM TEAB). Thermoanaerobacter tengcongensis (T. tengcongensis). This After the labeling reaction was kept at room temperature for 2 h, 10 μL of 5% ethanolamine dissolved in water was added to thermophilic bacterium, which was first isolated from China quench the reaction. The solvent was removed by Speedvac, and comprehensively studied at its genomics and proteomics, is and the labeled peptides were ready for LC-MS/MS. an ideal model in exploring the molecular mechanisms of The 4-plex iTRAQ labeling
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