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