Application of Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) to Monitor Olfactory Proteomes During Alzheimer’s Disease Progression

  • Andrea González Morales
  • Mercedes Lachén-Montes
  • María Ibáñez-Vea
  • Enrique Santamaría
  • Joaquín Fernández-Irigoyen
Part of the Neuromethods book series (NM, volume 127)


Olfactory impairment is a common early feature in several neurodegenerative diseases, including Alzheimer’s disease (AD). One of the main brain regions involved in the processing of olfactory information is the olfactory bulb (OB). In this chapter, we describe the use of isobaric tags for relative and absolute quantification (iTRAQ) to study the OB proteome during the neurodegenerative process in AD subjects. These chemical tags label all peptides in a protein digest via free amines at the peptide N-terminus and on the side chain of lysine residues. Labeled samples are then pooled and analyzed simultaneously using mass spectrometry (MS). Since these tags are isobaric, the intensity of each peak is the sum of the intensity of the peptide from all samples, thus enhancing sensitivity in MS. Similarly, upon peptide fragmentation, amino acid sequence ions also show this summed intensity. However, the distinct distribution of isotopes in the tags is such that when the tags fragment, a tag-specific reporter ion is released. The relative amount of peptide in each of the labeled samples will be represented by the relative intensities of these ions. In summary, this chapter describes the experimental procedure followed to analyzed human OB samples from AD subjects with the aim to increase the understanding of the molecular mechanisms that underlie neurodegeneration in this brain region.

Key words

Olfactory bulb Neurodegeneration Alzheimer’s disease iTRAQ Quantitative proteomics 



This work was funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (Ref. SAF2014-59340-R), Department of Economic Development from Government of Navarra (Ref. PC025), and Obra Social la Caixa to E.S. A.G.M. is supported by PEJ-2014-A-61949 (MINECO). The proteomics unit of Navarrabiomed is a member of Proteored, PRB2-ISCIII, and is supported by grant PT13/0001 of the PE I+D+I 2013–2016 funded by ISCIII and FEDER.


  1. 1.
    Lobo A, Launer LJ, Fratiglioni L, Andersen K, Di Carlo A, Breteler MM, Copeland JR, Dartigues JF, Jagger C, Martinez-Lage J, Soininen H, Hofman A (2000) Prevalence of dementia and major subtypes in Europe: a collaborative study of population-based cohorts. Neurologic Diseases in the Elderly Research Group. Neurology 54(11 Suppl 5):S4–S9PubMedGoogle Scholar
  2. 2.
    Braak H, Braak E (1991) Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 82(4):239–259CrossRefPubMedGoogle Scholar
  3. 3.
    Doty RL (2008) The olfactory vector hypothesis of neurodegenerative disease: is it viable? Ann Neurol 63(1):7–15. doi: 10.1002/ana.21327 CrossRefPubMedGoogle Scholar
  4. 4.
    Attems J, Walker L, Jellinger KA (2014) Olfactory bulb involvement in neurodegenerative diseases. Acta Neuropathol 127(4):459–475. doi: 10.1007/s00401-014-1261-7 CrossRefPubMedGoogle Scholar
  5. 5.
    Wu WW, Wang G, Baek SJ, Shen RF (2006) Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI TOF/TOF. J Proteome Res 5(3):651–658. doi: 10.1021/pr050405o CrossRefPubMedGoogle Scholar
  6. 6.
    Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3(12):1154–1169. doi: 10.1074/mcp.M400129-MCP200. M400129-MCP200 [pii]CrossRefPubMedGoogle Scholar
  7. 7.
    Craft GE, Chen A, Nairn AC (2013) Recent advances in quantitative neuroproteomics. Methods 61(3):186–218. doi: 10.1016/j.ymeth.2013.04.008. S1046-2023(13)00112-6 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Zelaya MV, Perez-Valderrama E, de Morentin XM, Tunon T, Ferrer I, Luquin MR, Fernandez-Irigoyen J, Santamaria E (2015) Olfactory bulb proteome dynamics during the progression of sporadic Alzheimer’s disease: identification of common and distinct olfactory targets across Alzheimer-related co-pathologies. Oncotarget 6(37):39437–39456. doi: 10.18632/oncotarget.6254. 6254 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Thompson A, Schafer J, Kuhn K, Kienle S, Schwarz J, Schmidt G, Neumann T, Johnstone R, Mohammed AK, Hamon C (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75(8):1895–1904CrossRefPubMedGoogle Scholar
  10. 10.
    Choe L, D'Ascenzo M, Relkin NR, Pappin D, Ross P, Williamson B, Guertin S, Pribil P, Lee KH (2007) 8-Plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer’s disease. Proteomics 7(20):3651–3660. doi: 10.1002/pmic.200700316 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1(5):376–386CrossRefPubMedGoogle Scholar
  12. 12.
    Latosinska A, Vougas K, Makridakis M, Klein J, Mullen W, Abbas M, Stravodimos K, Katafigiotis I, Merseburger AS, Zoidakis J, Mischak H, Vlahou A, Jankowski V (2015) Comparative analysis of label-free and 8-Plex iTRAQ approach for quantitative tissue proteomic analysis. PLoS One 10(9):e0137048. doi: 10.1371/journal.pone.0137048. PONE-D-15-05425 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Wang H, Alvarez S, Hicks LM (2012) Comprehensive comparison of iTRAQ and label-free LC-based quantitative proteomics approaches using two Chlamydomonas reinhardtii strains of interest for biofuels engineering. J Proteome Res 11(1):487–501. doi: 10.1021/pr2008225 CrossRefPubMedGoogle Scholar
  14. 14.
    Houel S, Abernathy R, Renganathan K, Meyer-Arendt K, Ahn NG, Old WM (2010) Quantifying the impact of chimera MS/MS spectra on peptide identification in large-scale proteomics studies. J Proteome Res 9(8):4152–4160. doi: 10.1021/pr1003856 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Michalski A, Cox J, Mann M (2011) More than 100,000 detectable peptide species elute in single shotgun proteomics runs but the majority is inaccessible to data-dependent LC-MS/MS. J Proteome Res 10(4):1785–1793. doi: 10.1021/pr101060v CrossRefPubMedGoogle Scholar
  16. 16.
    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. doi: 10.1021/pr900634c CrossRefPubMedGoogle Scholar
  17. 17.
    Karp NA, Huber W, Sadowski PG, Charles PD, Hester SV, Lilley KS (2010) Addressing accuracy and precision issues in iTRAQ quantitation. Mol Cell Proteomics 9(9):1885–1897. doi: 10.1074/mcp.M900628-MCP200. M900628-MCP200 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Ow SY, Salim M, Noirel J, Evans C, Wright PC (2011) Minimising iTRAQ ratio compression through understanding LC-MS elution dependence and high-resolution HILIC fractionation. Proteomics 11(11):2341–2346. doi: 10.1002/pmic.201000752 CrossRefPubMedGoogle Scholar
  19. 19.
    Ting L, Rad R, Gygi SP, Haas W (2011) MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat Methods 8(11):937–940. doi: 10.1038/nmeth.1714. nmeth.1714 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Wenger CD, Lee MV, Hebert AS, McAlister GC, Phanstiel DH, Westphall MS, Coon JJ (2011) Gas-phase purification enables accurate, multiplexed proteome quantification with isobaric tagging. Nat Methods 8(11):933–935. doi: 10.1038/nmeth.1716. nmeth.1716 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    McAlister GC, Huttlin EL, Haas W, Ting L, Jedrychowski MP, Rogers JC, Kuhn K, Pike I, Grothe RA, Blethrow JD, Gygi SP (2012) Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses. Anal Chem 84(17):7469–7478. doi: 10.1021/ac301572t CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Thingholm TE, Palmisano G, Kjeldsen F, Larsen MR (2010) Undesirable charge-enhancement of isobaric tagged phosphopeptides leads to reduced identification efficiency. J Proteome Res 9(8):4045–4052. doi: 10.1021/pr100230q CrossRefPubMedGoogle Scholar
  23. 23.
    Andrews GL, Simons BL, Young JB, Hawkridge AM, Muddiman DC (2011) Performance characteristics of a new hybrid quadrupole time-of-flight tandem mass spectrometer (TripleTOF 5600). Anal Chem 83(13):5442–5446. doi: 10.1021/ac200812d CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Bradshaw RA, Burlingame AL, Carr S, Aebersold R (2006) Reporting protein identification data: the next generation of guidelines. Mol Cell Proteomics 5(5):787–788. doi: 10.1074/mcp.E600005-MCP200. 5/5/787 [pii]CrossRefPubMedGoogle Scholar
  25. 25.
    Unwin RD, Griffiths JR, Whetton AD (2010) Simultaneous analysis of relative protein expression levels across multiple samples using iTRAQ isobaric tags with 2D nano LC-MS/MS. Nat Protoc 5(9):1574–1582. doi: 10.1038/nprot.2010.123. nprot.2010.123 [pii]CrossRefPubMedGoogle Scholar
  26. 26.
    Scheerlinck E, Dhaenens M, Van Soom A, Peelman L, De Sutter P, Van Steendam K, Deforce D (2015) Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry. Anal Biochem 490:14–19. doi: 10.1016/j.ab.2015.08.018. S0003-2697(15)00394-2 [pii]CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Andrea González Morales
    • 1
  • Mercedes Lachén-Montes
    • 1
  • María Ibáñez-Vea
    • 2
  • Enrique Santamaría
    • 1
  • Joaquín Fernández-Irigoyen
    • 1
  1. 1.Clinical Neuroproteomics Unit, Navarrabiomed, Navarra Health DepartmentPublic University of Navarra, Proteored-Institute of Health Carlos III (ISCIII), Navarra Institute for Health Research (IdiSNA)PamplonaSpain
  2. 2.Immunomodulation Group, Navarrabiomed Biomedical Research CenterNavarra Institute for Health Research (IdiSNA)PamplonaSpain

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