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Quantitative proteomic analysis of LPS-induced differential immune response associated with TLR4 Polymorphisms by multiplex amino acid coded mass tagging.

ABSTRACT: Polymorphisms at toll-like receptor 4 (TLR4) gene have been found to be associated with immune disorders. A murine macrophage cell line GG2EE derived from C3H/HeJ mice with a polymorphism site at TLR4 is hyposensitive to lipopolysaccharide (LPS). To study the molecular base of diverse TLR4-mediated immune responses, the proteomic changes in both TLR4-deficient and wild-type cell lines in response to the same LPS challenge were quantitatively compared by using multiplex amino acid coded mass tagging (AACT)/SILAC-assisted MS. This strategy allows encoding of two distinct cell populations with different stable isotope-tagged lysine residues as the "in-spectra" quantitative markers. In MS analysis of tryptic peptides derived from the equally mixed three cell populations, the lysine-containing peptides originated from two LPS-stimulated cell populations can be clearly distinguished by their different mass shifts from the unstimulated and unlabeled counterpart. The LPS-induced differential protein expression in TLR4-deficient and wild-type proteomes were obtained by comparing the intensities of isotopically encoded peptides. Among the more than 900 proteins identified, 35 were found to be deregulated at different levels in these two cell lines stimulated by LPS. This multiplex mass-tagging methodology can be readily extended to other comparative proteomic quantitation of different cell populations.

SUBMITTER: Gu S

PROVIDER: S-EPMC4302329 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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Quantitative proteomic analysis of LPS-induced differential immune response associated with TLR4 Polymorphisms by multiplex amino acid coded mass tagging.

Gu Sheng S Wang Tianyi T Chen Xian X

Proteomics 20080801 15

Polymorphisms at toll-like receptor 4 (TLR4) gene have been found to be associated with immune disorders. A murine macrophage cell line GG2EE derived from C3H/HeJ mice with a polymorphism site at TLR4 is hyposensitive to lipopolysaccharide (LPS). To study the molecular base of diverse TLR4-mediated immune responses, the proteomic changes in both TLR4-deficient and wild-type cell lines in response to the same LPS challenge were quantitatively compared by using multiplex amino acid coded mass tagg ...[more]

PMID: 18654986

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Project description:Peroxidation of cellular membrane lipids, rich in polyunsaturated fatty acids, generates electrophilic, ?, ?-unsaturated aldehydes such as 4-hydroxy-2-nonenal (HNE). HNE is a highly reactive and cytotoxic molecule that can react with the nucleophilic sites in proteins causing posttranslational modification. The identification of protein targets is an important first step; however, quantitative profiling of site-specific modifications is necessary to understand the biological impact of HNE-induced carbonylation. We report a method that uses light (H(12)CHO) and heavy (D(13)CDO) isotopic variant of formaldehyde to differentially label primary amines (N-termini and ?-amino group of lysines) in peptides through reductive methylation and, combined with selective enrichment of modified peptides, permits comparison of the extent of carbonylation in two samples after mixing for simultaneous liquid chromatography-mass spectrometry. Specifically, dimethyl-labeled peptide carbonyls were fractionated from unmodified peptides using solid-phase hydrazide chemistry to immobilize them to porous glass beads and, after removing the unmodified peptides by thoroughly washing the beads, subsequently recover them by acid-catalyzed hydrolysis. The method was developed using HNE-modified synthetic peptides and also showing enrichment from a complex matrix of digested human plasma proteins. Applicability was confirmed using apomyoglobin as an analyte, implicating thereby its potential value to proteome-wide identification and relative quantification of posttranslational protein carbonylation with residue-specific information. Because HNE attachment may not necessarily cause change in protein abundance, this modification-focused quantification should facilitate the characterization of accompanied changes in protein function and, also, provide important insights into molecular signaling mechanisms and a better understanding of cellular processes associated with oxidative stress.

Dataset Information

Quantitative proteomic analysis of LPS-induced differential immune response associated with TLR4 Polymorphisms by multiplex amino acid coded mass tagging.

Publications

Quantitative proteomic analysis of LPS-induced differential immune response associated with TLR4 Polymorphisms by multiplex amino acid coded mass tagging.

Similar Datasets

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