A proteomic method for the analysis of changes in protein concentrations in response to systemic perturbations using metabolic incorporation of stable isotopes and mass spectrometry

Gustavsson, Niklas; Greber, Boris; Kreitler, Thomas; Himmelbauer, Heinz; Lehrach, Hans; Gobom, Johan

doi:10.1002/pmic.200401193

Cited by 32 publications

(19 citation statements)

References 17 publications

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“…They do also not reflect the % of 15 N incorporation (57, 58), a complex calculation process that is not available in an automated manner and thus not implemented here. Nevertheless, RIA also called q-value has often been used as the basis for turnover calculations (59, 60). Here, we used RIAs, data on 14 N and 15 N abundances and compared changes over time as well as protein fold changes for the estimation of relative turnover dynamics.…”

Section: Resultsmentioning

confidence: 99%

Drought and Recovery: Independently Regulated Processes Highlighting the Importance of Protein Turnover Dynamics and Translational Regulation in Medicago truncatula

Lyon

Castillejo

Mehmeti-Tershani

et al. 2016

Molecular & Cellular Proteomics

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Climate change in conjunction with population growth necessitates a systems biology approach to characterize plant drought acclimation as well as a more thorough understanding of the molecular mechanisms of stress recovery. Plants are exposed to a continuously changing environment. Extremes such as several weeks of drought are followed by rain. This requires a molecular plasticity of the plant enabling drought acclimation and the necessity of deacclimation processes for recovery and continuous growth.During drought stress and subsequent recovery, the metabolome and proteome are regulated through a sequence of molecular processes including synthesis and degradation and molecular interaction networks are part of this regulatory process. In order to study this complex regulatory network, a comprehensive analysis is presented for the first time, investigating protein turnover and regulatory classes of proteins and metabolites during a stress recovery scenario in the model legume Medicago truncatula. The data give novel insights into the molecular capacity and differential processes required for acclimation and deacclimation of severe drought stressed plants.Functional cluster and network analyses unraveled independent regulatory mechanisms for stress and recovery with different dynamic phases that during the course of recovery define the plants deacclimation from stress. The combination of relative abundance levels and turnover analysis revealed an early transition phase that seems key for recovery initiation through water resupply and is independent from renutrition. Thus, a first indication for a metabolite and protein-based load capacity was observed necessary for the recovery from drought, an important but thus far ignored possible feature toward tolerance. The data indicate that apart from the plants molecular stress response mechanisms, plasticity may be related to the nutritional status of the plant prior to stress initiation. A new perspective and possible new targets as well as metabolic mechanisms for future plant-bioengineering toward enhanced drought stress tolerance are presented.

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Section: Resultsmentioning

confidence: 99%

Drought and Recovery: Independently Regulated Processes Highlighting the Importance of Protein Turnover Dynamics and Translational Regulation in Medicago truncatula

Lyon

Castillejo

Mehmeti-Tershani

et al. 2016

Molecular & Cellular Proteomics

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“…399, 1296.684, 1619.822, and 2093.086 D was used. The relative amount of newly synthesized protein after the 15 N pulse was calculated according to Gustavsson et al (2005). T. elongatus cells were harvested 24 h after the 15 N pulse, followed by preparation of PSII M(low) and SDS-PAGE analysis.…”

Section: Mass Spectrometrymentioning

confidence: 99%

Psb27, a Cyanobacterial Lipoprotein, Is Involved in the Repair Cycle of Photosystem II

Nowaczyk

Hebeler

Schlodder³

et al. 2006

Plant Cell

171

201

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Photosystem II (PSII) performs one of the key reactions on our planet: the light-driven oxidation of water. This fundamental but very complex process requires PSII to act in a highly coordinated fashion. Despite detailed structural information on the fully assembled PSII complex, the dynamic aspects of formation, processing, turnover, and degradation of PSII with at least 19 subunits and various cofactors are still not fully understood. Transient complexes are especially difficult to characterize due to low abundance, potential heterogeneity, and instability. Here, we show that Psb27 is involved in the assembly of the water-splitting site of PSII and in the turnover of the complex. Psb27 is a bacterial lipoprotein with a specific lipid modification as shown by matrix-assisted laser-desorption ionization time of flight mass spectrometry. The combination of HPLC purification of four different PSII subcomplexes and 15 N pulse label experiments revealed that lipoprotein Psb27 is part of a preassembled PSII subcomplex that represents a distinct intermediate in the repair cycle of PSII.

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“…The use of amino acids labeled with stable isotopes rather than radioisotopes is a solution that is applicable to a mass spectrometry-based proteome-wide approach (3)(4)(5)(6)(7). However, this method needs extensive labeling times as the unlabeled bulk of the protein content of the cell will also be detected.…”

mentioning

confidence: 99%

Identification and Quantitation of Newly Synthesized Proteins in Escherichia coli by Enrichment of Azidohomoalanine-labeled Peptides with Diagonal Chromatography

Kramer¹,

Sprenger

Back³

et al. 2009

Molecular & Cellular Proteomics

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A method is presented to identify and quantify several hundreds of newly synthesized proteins in Escherichia coli upon pulse labeling cells with the methionine analogue azidohomoalanine (azhal). For the first 30 min after inoculation, a methionine-auxotrophic strain grows equally well on azhal as on methionine. Upon a pulse of 15 min and digestion of total protein, azhallabeled peptides are isolated by a retention time shift between two reversed phase chromatographic runs. The retention time shift is induced by a reaction selective for the azido group in labeled peptides using tris(2-carboxyethyl)phosphine. Selectively Knowledge about protein synthesis and degradation rates on a proteome-wide scale is an important requirement for advanced modeling of the kinetics of cellular response networks. Pulse-chase labeling with radiolabeled compounds combined with separation of proteins by two-dimensional gel electrophoresis has already been applied (1, 2). However, this approach has drawbacks, such as difficulties to detect very acidic, basic, or hydrophobic proteins (e.g. membrane proteins). The possible occurrence of more than one protein in a gel spot, masking the relative contribution of each species to the total radioactivity, is another intrinsic difficulty.The use of amino acids labeled with stable isotopes rather than radioisotopes is a solution that is applicable to a mass spectrometry-based proteome-wide approach (3-7). However, this method needs extensive labeling times as the unlabeled bulk of the protein content of the cell will also be detected. Detection of small amounts of labeled, newly synthesized proteins in the presence of large amounts of unlabeled proteins is severely limited by the dynamic range of the mass spectrometer. This requirement for longer labeling times hampers identification and quantitation of transient changes in protein expression following perturbations upon pulse labeling. What is needed is an amino acid analogue that can be distinguished from its natural counterpart, can be used in a gel-free proteomics approach, and will facilitate the isolation of newly synthesized proteins from a large pool of pre-existing proteins. This will enhance identification and increase the dynamic range as well as the sensitivity of detection for transiently expressed proteins.In recent efforts, non-natural amino acids have been used to distinguish between newly synthesized proteins and preFrom ‡Mass Spectrometry of Biomacromolecules and **Molecular Microbial Physiology,

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A proteomic method for the analysis of changes in protein concentrations in response to systemic perturbations using metabolic incorporation of stable isotopes and mass spectrometry

Cited by 32 publications

References 17 publications

Drought and Recovery: Independently Regulated Processes Highlighting the Importance of Protein Turnover Dynamics and Translational Regulation in Medicago truncatula

Drought and Recovery: Independently Regulated Processes Highlighting the Importance of Protein Turnover Dynamics and Translational Regulation in Medicago truncatula

Psb27, a Cyanobacterial Lipoprotein, Is Involved in the Repair Cycle of Photosystem II

Identification and Quantitation of Newly Synthesized Proteins in Escherichia coli by Enrichment of Azidohomoalanine-labeled Peptides with Diagonal Chromatography

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