Quantitative Tracking of Isotope Flows in Proteomes of Microbial Communities

Pan, Chongle; Fischer, Curt R.; Hyatt, Doreene R.; Bowen, Benjamin P.; Hettich, Robert L.; Banfield, Jillian F.

doi:10.1074/mcp.m110.006049

Cited by 73 publications

(99 citation statements)

References 32 publications

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“…For example, when labeled substrates are added at the same time as changes in a physiological state, it will be possible to analyze the expression pattern by analyzing protein-specific incorporation. A similar use of stable isotope labels in a protein SIP experiment has been proposed (10), but the peptide MS/MS approach for amino acid SIP described here can provide incorporation data without the need for massive amounts of protein data as shown for determination via a decimal place method (9), or for extensive computer processing hours when calculated via a stepwise comparison with hypothetical incorporation percentages (18). Similarly to protein SIP (25), turnover rates of proteins can be analyzed.…”

Section: Cultivationmentioning

confidence: 99%

“…Our approach, here called the peptide MS/MS ap-Waters, Eschborn, Germany) with water containing 0.1% formic acid at a flow rate of 15 l min Ϫ1 . After 6 min, the peptides were eluted onto the separation column (nanoAcquity UPLC column, C 18 , 75 m by 100 mm by 1.7 m; Waters, Eschborn, Germany). Chromatography was performed by using 0.1% formic acid in solvents A (100% water) and B (100% acetonitrile), with peptides eluted over 90 min with a 6 to 40% solvent B gradient using a nano-high-pressure liquid chromatography (nano-HPLC) system (nanoAcquity; Waters, Eschborn, Germany) coupled to an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany).…”

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confidence: 99%

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Stable Isotope Peptide Mass Spectrometry To Decipher Amino Acid Metabolism in Dehalococcoides Strain CBDB1

et al. 2012

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cDehalococcoides species are key players in the anaerobic transformation of halogenated solvents at contaminated sites. Here, we analyze isotopologue distributions in amino acid pools from peptides of Dehalococcoides strain CBDB1 after incubation with 13 C-labeled acetate or bicarbonate as a carbon source. The resulting data were interpreted with regard to genome annotations to identify amino acid biosynthesis pathways. In addition to using gas chromatography-mass spectrometry (GC-MS) for analyzing derivatized amino acids after protein hydrolysis, we introduce a second, much milder method, in which we directly analyze peptide masses after tryptic digest and peptide fragments by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With this method, we identify isotope incorporation patterns for 17 proteinaceous amino acids, including proline, cysteine, lysine, and arginine, which escaped previous analyses in Dehalococcoides. Our results confirmed lysine biosynthesis via the ␣-aminoadipate pathway, precluding lysine formation from aspartate. Similarly, the isotopologue pattern obtained for arginine provided biochemical evidence of its synthesis from glutamate. Direct peptide MS/MS analysis of the labeling patterns of glutamine and asparagine, which were converted to glutamate and aspartate during protein hydrolysis, gave biochemical evidence of their precursors and confirmed glutamate biosynthesis via a Re-specific citrate synthase. By addition of unlabeled free amino acids to labeled cells, we show that in strain CBDB1 none of the 17 tested amino acids was incorporated into cell mass, indicating that they are all synthesized de novo. Our approach is widely applicable and provides a means to analyze amino acid metabolism by studying specific proteins even in mixed consortia. Dehalococcoides species are strictly anaerobic bacteria known for the ability to use a variety of halogenated aliphatic and aromatic compounds as respiratory electron acceptors. Many of these organohalides are persistent and toxic groundwater pollutants. Dehalococcoides isolates use hydrogen as the sole electron donor and acetate plus bicarbonate as carbon sources. While some biochemical details of the respiratory electron chain have been studied, knowledge of the carbon metabolism of Dehalococcoides spp. is scarce. Sequenced and annotated genomes of several Dehalococcoides strains provide a basis for the generation of hypotheses for carbon metabolism but also highlight gaps in our understanding (12,15,21). For instance, the genome annotations lack key genes for the biosynthesis of methionine, alanine, serine, glycine, and threonine. In addition, genes may be annotated incorrectly as evidenced by the recent identification of a gene encoding a Recitrate synthase previously annotated as homocitrate synthase (14). A modeling approach has used a pan-genome of all available Dehalococcoides sequences to develop a model for the central metabolism and growth of Dehalococcoides species (2). This study emphasi...

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Section: Cultivationmentioning

confidence: 99%

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confidence: 99%

Stable Isotope Peptide Mass Spectrometry To Decipher Amino Acid Metabolism in Dehalococcoides Strain CBDB1

et al. 2012

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“…The in situ study of microbial activity in natural and engineered ecosystems is therefore of great interest. For this purpose, several elegant methods have been established that use either transcriptional or translational activity of community members (i.e., metatranscriptomics, metaproteomics) (1)(2)(3) or the incorporation of isotopically labeled substrates into biomolecules (4)(5)(6)(7)(8)(9)(10) to infer the ecophysiology of microbes in such systems. However, these bulk techniques do not offer sufficient spatial resolution to study microbial activities at the micrometer scale.…”

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confidence: 99%

Tracking heavy water (D ₂ O) incorporation for identifying and sorting active microbial cells

Berry

Mader

Lee

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

392

512

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Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D 2 O) combined with Raman microspectroscopy. Incorporation of D 2 O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscaleresolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D 2 O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Ramanbased cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/ or glucosamine were identified, demonstrating the potential of the nondestructive D 2 O-Raman approach for targeted sorting of microbial cells with defined functional properties for singlecell genomics.ecophysiology | single-cell microbiology | carbohydrate utilization | nitrifier | Raman microspectroscopy M icroorganisms play a vital role in many environments. They mediate global biogeochemical cycles, catalyze biotechnological processes, and contribute to health and disease in the human body. The in situ study of microbial activity in natural and engineered ecosystems is therefore of great interest. For this purpose, several elegant methods have been established that use either transcriptional or translational activity of community members (i.e., metatranscriptomics, metaproteomics) (1-3) or the incorporation of isotopically labeled substrates into biomolecules (4-10) to infer the ecophysiology of microbes in such systems. However, these bulk techniques do not offer sufficient spatial resolution to study microbial activities at the micrometer scale. Therefore, important information can be overlooked because microbial communities are frequently spatially structured (e.g., biofilms) (11) and contain populations with life cycles (12,13). Furthermore, even apparently identical cells in clonal populations can have strongly divergent activities (14).Consequently, microbial ecophysiology is ideally studied also at the level of the single cell, but only a restricted number of approaches exist for determining physiological properties of individual cells in a microbial community. For exa...

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“…This method has been described using 15 N (Snijders et al, 2005;Pan et al, 2011) and also 36 S . The incorporation of stable isotopes into proteins by metabolic labeling can be used to quantify protein abundances and to observe changes in the microbial community structure.…”

Section: Metabolic Labelingmentioning

confidence: 99%

Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology

et al. 2013

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The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes ( 13 C, 15 N, 36 S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying the carbon flux within microbial consortia. Furthermore, the combination of protein-SIP with established tools in microbial ecology such as other stable isotope probing techniques are discussed.

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Quantitative Tracking of Isotope Flows in Proteomes of Microbial Communities

Cited by 73 publications

References 32 publications

Stable Isotope Peptide Mass Spectrometry To Decipher Amino Acid Metabolism in Dehalococcoides Strain CBDB1

Stable Isotope Peptide Mass Spectrometry To Decipher Amino Acid Metabolism in Dehalococcoides Strain CBDB1

Tracking heavy water (D ₂ O) incorporation for identifying and sorting active microbial cells

Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology

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Quantitative Tracking of Isotope Flows in Proteomes of Microbial Communities

Cited by 73 publications

References 32 publications

Stable Isotope Peptide Mass Spectrometry To Decipher Amino Acid Metabolism in Dehalococcoides Strain CBDB1

Stable Isotope Peptide Mass Spectrometry To Decipher Amino Acid Metabolism in Dehalococcoides Strain CBDB1

Tracking heavy water (D 2 O) incorporation for identifying and sorting active microbial cells

Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology

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Tracking heavy water (D ₂ O) incorporation for identifying and sorting active microbial cells