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Metabolite profiles of human immunodeficiency virus infected CD4+ T cells and macrophages using LC-MS/MS analysis.

ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) infects both activated CD4+ T cells and macrophages. We tested if liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology can monitor metabolic alterations induced by HIV-1 in the infected cells. Here we monitored glucose uptake and conducted LC-MS/MS-based metabolomic analysis in HIV-1 infected primary human CD4+ T cells and a macrophage model system: differentiated U1 (HIV-1 producing) and differentiated U937 (control) cells. HIV-1 infected CD4+ T cells have higher glucose uptake and increases in several metabolite pool sizes, whereas HIV-1 producing macrophages had substantial reductions in glucose uptake and steady state glycolytic intermediates. This data suggests that the two HIV-1 target cell types exhibit very different metabolic outcomes during viral production. This study also validates the LC-MS/MS technology as an effective metabolomic approach to monitor various metabolic alterations made by HIV-1 infection.

SUBMITTER: Hollenbaugh JA

PROVIDER: S-EPMC3107887 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Metabolite profiles of human immunodeficiency virus infected CD4+ T cells and macrophages using LC-MS/MS analysis.

Hollenbaugh Joseph A JA Munger Joshua J Kim Baek B

Virology 20110511 2

Human immunodeficiency virus type 1 (HIV-1) infects both activated CD4+ T cells and macrophages. We tested if liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology can monitor metabolic alterations induced by HIV-1 in the infected cells. Here we monitored glucose uptake and conducted LC-MS/MS-based metabolomic analysis in HIV-1 infected primary human CD4+ T cells and a macrophage model system: differentiated U1 (HIV-1 producing) and differentiated U937 (control) cells. HIV-1 infec ...[more]

PMID: 21565377

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Project description:BackgroundThe observed molecular weight of a protein on a 1D polyacrylamide gel can provide meaningful insight into its biological function. Differences between a protein's observed molecular weight and that predicted by its full length amino acid sequence can be the result of different types of post-translational events, such as alternative splicing (AS), endoproteolytic processing (EPP), and post-translational modifications (PTMs). The characterization of these events is one of the important goals of total proteome profiling (TPP). LC/MS/MS has emerged as one of the primary tools for TPP, but since this method identifies tryptic fragments of proteins, it has not generally been used for large-scale determination of the molecular weight of intact proteins in complex mixtures.ResultsWe have developed a set of computational tools for extracting molecular weight information of intact proteins from total proteome profiles in a high throughput manner using 1D-PAGE and LC/MS/MS. We have applied this technology to the proteome profile of a human lymphoblastoid cell line under standard culture conditions. From a total of 1 x 10(7) cells, we identified 821 proteins by at least two tryptic peptides. Additionally, these 821 proteins are well-localized on the 1D-SDS gel. 656 proteins (80%) occur in gel slices in which the observed molecular weight of the protein is consistent with its predicted full-length sequence. A total of 165 proteins (20%) are observed to have molecular weights that differ from their predicted full-length sequence. We explore these molecular-weight differences based on existing protein annotation.ConclusionWe demonstrate that the determination of intact protein molecular weight can be achieved in a high-throughput manner using 1D-PAGE and LC/MS/MS. The ability to determine the molecular weight of intact proteins represents a further step in our ability to characterize gene expression at the protein level. The identification of 165 proteins whose observed molecular weight differs from the molecular weight of the predicted full-length sequence provides another entry point into the high-throughput characterization of protein modification.

Dataset Information

Metabolite profiles of human immunodeficiency virus infected CD4+ T cells and macrophages using LC-MS/MS analysis.

Publications

Metabolite profiles of human immunodeficiency virus infected CD4+ T cells and macrophages using LC-MS/MS analysis.

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