Abstract
The Applied Biosystems iTRAQ technique is based on derivatization of peptide samples with mass-balanced tags. The samples are then combined, and quantitation is based on reporter ion abundances in the MS/MS spectrum. The iTRAQ technique, with its capability for the analysis of either 4 or 8 samples in a single LC-MS/MS analysis, is one of the most powerful techniques available today for “shotgun” quantitative proteomics. However, it is not an easy technique, and involves multiple – and critical – digestion, separation, and derivatization steps. Our laboratory has spent several years developing over 40 standard operating procedures (SOPs) for the generation of reproducible iTRAQ data. These protocols are designed for generating and analyzing iTRAQ data on the Applied Biosystems 4800 MALDI-TOF/TOF instrument and the Applied Biosystems QSTAR ESI-MS/MS, which are two of the most commonly-used instruments for iTRAQ analysis. We have also included sections on the Applied Biosystems ProteinPilot software which is used for analyzing the data and obtaining protein expression ratios. Standard operating procedures are obviously designed to be used with these specific instruments. For laboratories with other instruments, we have tried to include sufficient annotation and descriptions of the procedures so that these protocols can be adapted for use on other equipment. Likewise, many of these protocols are not specific to iTRAQ, and can be used for sample preparation and analysis for other assays as well. For more complete and detailed protocols, the reader is referred to the on-line supplementary information on our website at http://www.proteincentre.com/itraq-book-chapter-supplemental-information.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Aguilar-Mahecha, A., Buchanan, M., Kuzyk, M.A., Borchers, C.H., and Basik, M. (2011). Comparison of blood collection tubes and processing protocols for plasma proteomics studies. Manuscript in preparation.
Alves, S., Fournier, F., Afonso, C., Wind, F., and Tabet, J.-C. (2006). Gas-phase ionization/desolvation processes and their effect on protein charge state distribution under matrix-assisted laser desorption/ionization conditions. Eur J Mass Spectrom 12, 369–383.
Anderson, N.L., and Anderson, N.G. (2002). The human plasma proteome: History, character, and diagnostic prospects. Mol Cell Proteomics 1, 845–867.
Applied_Biosystems (2004a). Analyst QS/BioAnalyst Tutorial.
Applied_Biosystems (2004b). iTRAQ™ Reagents Amine-Modifying Labeling Reagents for Multiplexed Relative and Absolute Protein Quantification Chemistry Reference Guide Part Number 4351918, Rev. A.
Applied_Biosystems (2004c). iTRAQ™ Reagents Amine-Modifying Labeling Reagents for Multiplexed Relative and Absolute Protein Quantitation Protocol, Rev. C. http://wwwabsciexcom/LITERATURE/cms_041463pdf.
Applied_Biosystems (2007). iTRAQ™ Reagents-8plex Amine-Modifying Labeling Reagents for Multiplexed Relative and Absolute Protein Quantitation Protocol Guide Part Number 4375249, Rev. B.
Applied_Biosystems/MDS_SCIEX (2001a). Analyst QS Getting Started Manual Part Number 1001933 B August 2001.
Applied_Biosystems/MDS_SCIEX (2001b). API QSTAR Pulsar I Hardware Manual, Part Number 1001553 B April 2001.
Applied_Biosystems/MDS_SCIEX (2005). 4800 MALDI TOF/TOF Analyzer Getting Started Guide Part Number 4352078 Rev. C April 2005.
Chang, W.C., Huang, L.C.L., Wang, Y.-S., Peng, W.-P., Chang, H.C., Hsu, N.Y., Yang, W.B., and Chen, C.H. (2007). Matrix-assisted laser desorption/ionization (MALDI) mechanism revisited. Anal Chim Acta 582, 1–9.
DeSouza, L.V., Grigull, J., Ghanny, S., Dube, V., Romaschin, A.D., Colgan, T.J., and Siu, K.W.M. (2007). Endometrial carcinoma biomarker discovery and verification using differentially tagged clinical samples with multidimensional liquid chromatography and tandem mass spectrometry. Mol Cell Proteomics 6, 1170–1182.
DeSouza, L.V., Romaschin, A.D., Colgan, T.J., and Siu, K.W.M. (2009). Absolute quantification of potential cancer markers in clinical tissue homogenates using multiple reaction monitoring on a hybrid triple quadrupole/linear ion trap tandem mass spectrometer. Anal Chem 81, 3462–3470.
Dionex/LC_Packings (2001a). Famos Autosampler for Capillary- and Nano HPLC User’s Manual. Part Number 160557.
Dionex/LC_Packings (2001b). Switchos Advanced Microcolumn Switching Device User’s Manual. Part Number 162013.
Dionex/LC_Packings (2002). Micro Fraction Collector User’s Manual. Part Number 161403.
Dionex/LC_Packings (2003). UltiMate Capillary and Nano HPLC System User’s Manual. Part Number 160534.
Eksigent (2004). Eksigent NanoLC User Manual is from 2004.
Eksigent (2005). Eksigent Nano1D Software User Guide.
Elliott, M., Smith, D., Kuzyk, M., Parker, C.E., and Borchers, C.H. (2009). Recent trends in quantitative proteomics. J Mass Spectrom 44, 1637–1660.
Gan, C.S., Chong, P.K., Pham, T.K., and Wright, P.C. (2007). Technical, experimental, and biological variations in isobaric tags for relative and absolute quantitation (iTRAQ). J Proteome Res 6, 821–827.
GE_Healthcare (2002). Äkta Prime User’s Manual; catalogue number: 18-1135-24, Edition AE.
Hardie, D., Jakubowski, P., Jackson, A., and Ohlund, L. (2006). Investigation of LC-MALDI Matrix Spotting for Improved TOF/TOF Analysis by Combined Spotting and Acquisition Strategies. Presented at the 54th ASMS Conference on Mass Spectrometry and Allied Topics, Seattle, WA, May 28–June 1, 2006.
Kuzyk, M.A., Ohlund, L.B., Elliott, M.H., Smith, D., Qian, H., Delaney, A., Hunter, C.L., and Borchers, C.H. (2009). A comparison of MS/MS-based, stable-isotope-labeled, quantitation performance on ESI-quadrupole TOF and MALDI-TOF/TOF mass spectrometers. Proteomics 9, 3328–3340.
Omenn, G.S., States, D.J., Adamski, M., Blackwell, T.W., Menon, R., Hermjakob, H., Apweiler, R., Haab, B.B., Simpson, R.J., Eddes, J.S., et al. (2005). Overview of the HUPO Plasma Proteome Project: Results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database. Proteomics 5, 3226–3245.
Ow, S.Y., Salim, M., Noirel, J., Evans, C., Rehman, I., and Wright, P.C. (2009). iTRAQ underestimation in simple and complex mixtures: “The Good, the Bad and the Ugly”. J Proteome Res 8, 5347–5355.
Parker, C.E., Warren, M.R., Loiselle, D.R., Dicheva, N.N., Scarlett, C.O., and Borchers, C.H. (2005). Identification of components of protein complexes. Methods Mol Biol 301, 117–151.
Proc, J.L., Kuzyk, M.A., Hardie, D.B., Yang, J., Smith, D.S., Jackson, A.M., Parker, C.E., and Borchers, C.H. (2010). A quantitative study of the effects of chaotropic agents, surfactants, and solvents on the digestion efficiency of human plasma proteins by trypsin. J Proteome Res 9, 5422–5437.
Proxeon_Biosystems (2002). Nano-Electrospray Installation and Operations Manual.
Ross, P.L., Huang, Y.N., Marchese, J.N., Williamson, B., Parker, K., Hattan, S., Khainovski, N., Pillai, S., Dey, S., Daniels, S., et al. (2004). Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3, 1154–1169.
Sigma-Aldrich (2005). BCA Protein Assay Kit Technical Bulletin. http://wwwsigmaaldrichcom/etc/medialib/docs/Sigma/Bulletin/qpbcabulPar0001Filetmp/qpbcabulpdf.
Sigma-Aldrich (2008). Seppro_column_user’s_guide. http://wwwsigmaaldrichcom/etc/medialib/docs/Sigma/Bulletin/sep030bulPar0001Filetmp/sep030bulpdf.
Simpson, R.J., Bernhard, O.K., Greening, D.W., and Moritz, R.L. (2008). Proteomics-driven cancer biomarker discovery: Looking to the future. Curr Opin Chem Biol 12, 72–77.
UVic_Proteomics_Center_SOPs (2010). http://www.proteincentre.com/services/standard-operating-procedures.
Wiese, S., Reidegeld, K.A., Meyer, H.E., and Warscheid, B. (2007). Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry in proteome research. Proteomics 7, 340–350.
Wolters, D.A., Washburn, M.P., and Yates, J.R., III (2001). An automated multidimensional protein identification technology for shotgun proteomics. Anal Chem 73, 5683–5690.
Acknowledgements
The authors would like to thank the organizations for funding work relevant to these methods including the PROOF Centre of Excellence, Genome Canada, Novartis, IBM, Genome BC, University of British Columbia VP Research, St. Paul’s Hospital Foundation, BC Transplant, and the James Hogg iCAPTURE Centre. We would also like to thank Drs. Steven Parnell and Oscar Alzate for critical reading of this manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media B.V.
About this chapter
Cite this chapter
Ohlund, L.B. et al. (2011). Standard Operating Procedures and Protocols for the Preparation and Analysis of Plasma Samples Using the iTRAQ Methodology. In: Ivanov, A., Lazarev, A. (eds) Sample Preparation in Biological Mass Spectrometry. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-0828-0_28
Download citation
DOI: https://doi.org/10.1007/978-94-007-0828-0_28
Published:
Publisher Name: Springer, Dordrecht
Print ISBN: 978-94-007-0758-0
Online ISBN: 978-94-007-0828-0
eBook Packages: Chemistry and Materials ScienceChemistry and Material Science (R0)