Abstract
Protein subcellular localization is a fundamental feature of posttranslational functional regulation. Traditional microscopy based approaches to study protein localization are typically of limited throughput, and dependent on the availability of antibodies with high specificity and sensitivity, or fluorescent fusion proteins. In this chapter we describe how Localization of Organelle Proteins by Isotope Tagging (LOPIT), a mass spectrometry based workflow coupling biochemical fractionation and iTRAQ™ 8-plex quantification, can be applied for the high-throughput characterization of protein localization in a mammalian cell culture line.
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Dunkley TP, Hester S, Shadforth IP, Runions J, Weimar T, Hanton SL et al (2006) Mapping the Arabidopsis organelle proteome. Proc Natl Acad Sci U S A 103(17):6518–6523
Sadowski PG, Dunkley TP, Shadforth IP, Dupree P, Bessant C, Griffin JL et al (2006) Quantitative proteomics approach to study subcellular localization of membrane proteins. Nat Protoc 1(4):1778–1789
Hall SL, Hester S, Griffin JL, Lilley KS, Jackson AP (2009) The organelle proteome of the DT40 lymphocyte cell line. Mol Cell Proteomics 8(6):1295–1305
Tan DJ, Dvinge H, Christoforou A, Bertone P, Martinez Arias A, Lilley KS (2009) Mapping organelle proteins and protein complexes in Drosophila melanogaster. J Proteome Res 8(6):2667–2678
de Duve C (1971) Tissue fraction-past and present. J Cell Biol 50(1):20
Kessner D, Chambers M, Burke R, Agus D, Mallick P (2008) ProteoWizard: open source software for rapid proteomics tools development. Bioinformatics 24(21):2534–2536
Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372
Lacerda CM, Xin L, Rogers I, Reardon KF (2008) Analysis of iTRAQ data using Mascot and Peaks quantification algorithms. Brief Funct Genomic Proteomic 7(2):119–126
Gatto L, Lilley KS (2012) MSnbase-an R/Bioconductor package for isobaric tagged mass spectrometry data visualization, processing and quantitation. Bioinformatics 28(2):288–289
Wang LH, Li DQ, Fu Y, Wang HP, Zhang JF, Yuan ZF et al (2007) pFind 2.0: a software package for peptide and protein identification via tandem mass spectrometry. Rapid Commun Mass Spectrom 21(18):2985–2991
Savitski MM, Mathieson T, Zinn N, Sweetman G, Doce C, Becher I et al (2013) Measuring and managing ratio compression for accurate iTRAQ/TMT quantification. J Proteome Res 12(8):3586–3598
Trotter MW, Sadowski PG, Dunkley TP, Groen AJ, Lilley KS (2010) Improved sub-cellular resolution via simultaneous analysis of organelle proteomics data across varied experimental conditions. Proteomics 10(23): 4213–4219
Gatto L, Breckels LM, Wieczorek S, Burger T, Lilley KS (2014) Mass-spectrometry-based spatial proteomics data analysis using pRoloc and pRolocdata. Bioinformatics (E-pub ahead of print)
Breckels LM, Gatto L, Christoforou A, Groen AJ, Lilley KS, Trotter MW (2013) The effect of organelle discovery upon sub-cellular protein localisation. J Proteomics 88:129–140
Ow SY, Salim M, Noirel J, Evans C, Rehman I, Wright PC (2009) iTRAQ underestimation in simple and complex mixtures: “the good, the bad and the ugly”. J Proteome Res 8(11): 5347–5355
Griffin TJ, Xie H, Bandhakavi S, Popko J, Mohan A, Carlis JV et al (2007) iTRAQ reagent-based quantitative proteomic analysis on a linear ion trap mass spectrometer. J Proteome Res 6(11):4200–4209
Evans C, Noirel J, Ow SY, Salim M, Pereira-Medrano AG, Couto N et al (2012) An insight into iTRAQ: where do we stand now? Anal Bioanal Chem 404(4):1011–1027
Christoforou AL, Lilley KS (2012) Isobaric tagging approaches in quantitative proteomics: the ups and downs. Anal Bioanal Chem 404(4):1029–1037
Ting L, Rad R, Gygi SP, Haas W (2011) MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat Methods 8(11):937–940
Wenger CD, Lee MV, Hebert AS, McAlister GC, Phanstiel DH, Westphall MS et al (2011) Gas-phase purification enables accurate, multiplexed proteome quantification with isobaric tagging. Nat Methods 8(11):933–935
Dayon L, Sonderegger B, Kussmann M (2012) Combination of gas-phase fractionation and MS3 acquisition modes for relative protein quantification with isobaric tagging. J Proteome Res 11(10):5081–5089
Acknowledgements
The authors would like to thank Daniel Nightingale and Julie Howard for their helpful comments to improve the clarity of the protocol. A.C. was funded by BBSRC grant BB/D526088/1.
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Christoforou, A., Arias, A.M., Lilley, K.S. (2014). Determining Protein Subcellular Localization in Mammalian Cell Culture with Biochemical Fractionation and iTRAQ 8-Plex Quantification. In: Martins-de-Souza, D. (eds) Shotgun Proteomics. Methods in Molecular Biology, vol 1156. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0685-7_10
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DOI: https://doi.org/10.1007/978-1-4939-0685-7_10
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