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Optimized proteomic analysis of a mouse model of cerebellar dysfunction using amine-specific isobaric tags.

ABSTRACT: Recent proteomic applications have demonstrated their potential for revealing the molecular mechanisms underlying neurodegeneration. The present study quantifies cerebellar protein changes in mice that are deficient in plasma membrane calcium ATPase 2 (PMCA2), an essential neuronal pump that extrudes calcium from cells and is abundantly expressed in Purkinje neurons. PMCA2-null mice display motor dyscoordination and unsteady gait deficits observed in neurological diseases such as multiple sclerosis and ataxia. We optimized an amine-specific isobaric tags (iTRAQ)-based shotgun proteomics workflow for this study. This workflow took consideration of analytical variance as a function of ion signal intensity and employed biological repeats to aid noise reduction. Even with stringent protein identification criteria, we could reliably quantify nearly 1000 proteins, including many neuronal proteins that are important for synaptic function. We identified 21 proteins that were differentially expressed in PMCA2-null mice. These proteins are involved in calcium homeostasis, cell structure and chromosome organization. Our findings shed light on the molecular changes that underlie the neurological deficits observed in PMCA2-null mice. The optimized workflow presented here will be valuable for others who plan to implement the iTRAQ method.

SUBMITTER: Hu J

PROVIDER: S-EPMC2553677 | biostudies-literature | 2006 Aug

REPOSITORIES: biostudies-literature

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Optimized proteomic analysis of a mouse model of cerebellar dysfunction using amine-specific isobaric tags.

Hu Jun J Qian Jin J Borisov Oleg O Pan Sanqiang S Li Yan Y Liu Tong T Deng Longwen L Wannemacher Kenneth K Kurnellas Michael M Patterson Christa C Elkabes Stella S Li Hong H

Proteomics 20060801 15

Recent proteomic applications have demonstrated their potential for revealing the molecular mechanisms underlying neurodegeneration. The present study quantifies cerebellar protein changes in mice that are deficient in plasma membrane calcium ATPase 2 (PMCA2), an essential neuronal pump that extrudes calcium from cells and is abundantly expressed in Purkinje neurons. PMCA2-null mice display motor dyscoordination and unsteady gait deficits observed in neurological diseases such as multiple sclero ...[more]

PMID: 16800037

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Project description:We report the development of novel reagents for cell-level protein quantification, referred to as Caltech isobaric tags (CITs), which offer several advantages in comparison with other isobaric tags (e.g., iTRAQ and TMT). Click chemistry, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), is applied to generate a gas-phase cleavable linker suitable for the formation of reporter ions. Upon collisional activation, the 1,2,3-triazole ring constructed by CuAAC participates in a nucleophilic displacement reaction forming a six-membered ring and releasing a stable cationic reporter ion. To investigate its utility in peptide mass spectrometry, the energetics of the observed fragmentation pathway are examined by density functional theory. When this functional group is covalently attached to a target peptide, it is found that the nucleophilic displacement occurs in competition with formation of b- and y-type backbone fragment ions regardless of the amino acid side chains present in the parent bioconjugate, confirming that calculated reaction energetics of reporter ion formation are similar to those of backbone fragmentations. Based on these results, we apply this selective fragmentation pathway for the development of CIT reagents. For demonstration purposes, duplex CIT reagent is prepared using a single isotope-coded precursor, allyl-d(5)-bromide, with reporter ions appearing at m/z 164 and 169. Isotope-coded allyl azides for the construction of the reporter ion group can be prepared from halogenated alkyl groups which are also employed for the mass balance group via N-alkylation, reducing the cost and effort for synthesis of isobaric pairs. Owing to their modular designs, an unlimited number of isobaric combinations of CIT reagents are, in principle, possible. The reporter ion mass can be easily tuned to avoid overlapping with common peptide MS/MS fragments as well as the low mass cutoff problems inherent in ion trap mass spectrometers. The applicability of the CIT reagent is tested with several model systems involving protein mixtures and cellular systems.

Dataset Information

Optimized proteomic analysis of a mouse model of cerebellar dysfunction using amine-specific isobaric tags.

Publications

Optimized proteomic analysis of a mouse model of cerebellar dysfunction using amine-specific isobaric tags.

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