An efficient protocol for the identification of protein phosphorylation in a seedless plant, sensitive enough to detect members of signalling cascades

Heintz, Dimitri; Wurtz, Virginie; High, Anthony A.; Dorsselaer, Alain Van; Reski, Ralf; Sarnighausen, Eric

doi:10.1002/elps.200305795

Cited by 47 publications

(64 citation statements)

References 45 publications

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“…In-gel digestion of proteins was performed with an automated protein digestion system, MassPREP Station (Micromass, Manchester, United Kingdom), as described previously (39). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) measurements was carried out with an Ultraflex TOF/TOF (Bruker Daltonik GmbH, Bremen, Germany), which was operated in the reflectron positive mode as previously described (18). Protein identification was made by peptide mass fingerprinting using the MASCOT program (Matrix Science, London, United Kingdom) and interrogation of different protein databases: the NCBI (National Center for Biotechnology) nonredundant protein database, SwissProt, and TrEMBL.…”

Section: Methodsmentioning

confidence: 99%

“…Protein identification was made by peptide mass fingerprinting using the MASCOT program (Matrix Science, London, United Kingdom) and interrogation of different protein databases: the NCBI (National Center for Biotechnology) nonredundant protein database, SwissProt, and TrEMBL. Additionally, protein identification was made by nanoscale capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described previously (18). Briefly, analysis of the digested proteins was performed using a CapLC capillary LC system (Waters) coupled to a hybrid quadrupole orthogonal acceleration time-of-flight tandem mass spectrometer, Q-TOF II (Waters).…”

Section: Methodsmentioning

confidence: 99%

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Genes, Enzymes, and Regulation of para -Cresol Metabolism in Geobacter metallireducens

Peters

Heintz²,

Johannes

et al. 2007

J Bacteriol

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In aerobic and facultatively anaerobic bacteria, the degradation of para-cresol (p-cresol) involves the initial hydroxylation to p-hydroxybenzyl alcohol by water catalyzed by the soluble, periplasmatic flavocytochrome p-cresol methylhydroxylase (PCMH; ␣ 2 ␤ 2 composition). In denitrifying bacteria the further metabolism proceeds via oxidation to p-hydroxybenzoate, the formation of p-hydroxybenzoyl-coenzyme A (CoA), and the subsequent dehydroxylation of the latter to benzoyl-CoA by reduction. In contrast, the strictly anaerobic Desulfobacterium cetonicum degrades p-cresol by addition to fumarate, yielding p-hydroxybenzylsuccinate. In this work, in vitro enzyme activity measurements revealed that the obligately anaerobic Geobacter metallireducens uses the p-cresol degradation pathway of denitrifying bacteria. Surprisingly, PCMH, which is supposed to catalyze both p-cresol hydroxylation and p-hydroxybenzyl alcohol oxidation to the corresponding aldehyde, was located in the membrane fraction. The ␣ subunit of the enzyme was present in two isoforms, suggesting an ␣␣␤ 2 composition. We propose that the unusual asymmetric architecture and the membrane association of PCMH might be important for alternative electron transfer routes to either cytochrome c (in the case of p-cresol oxidation) or to menaquinone (in the case of p-hydroxybenzyl alcohol oxidation). Unusual properties of further enzymes of p-cresol metabolism, p-hydroxybenzoate-CoA ligase, and p-hydroxybenzoyl-CoA reductase were identified and are discussed. A proteomic approach identified a gene cluster comprising most of the putative structural genes for enzymes involved in p-cresol metabolism (pcm genes). Reverse transcription-PCR studies revealed a different regulation of transcription of pcm genes and the corresponding enzyme activities, suggesting the presence of posttranscriptional regulatory elements.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Genes, Enzymes, and Regulation of para -Cresol Metabolism in Geobacter metallireducens

Peters

Heintz²,

Johannes

et al. 2007

J Bacteriol

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“…Among the few examples, phosphoproteins of Arabidopsis plasma membranes (23) and phosphoproteins in the moss Physcomitrella patens (9) have been characterized. Here, we have enriched phosphoproteins from C. reinhardtii by using IMAC in combination with Ga 3ϩ as the metal component.…”

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confidence: 99%

Analysis of the Phosphoproteome of Chlamydomonas reinhardtii Provides New Insights into Various Cellular Pathways

et al. 2006

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The unicellular flagellated green alga Chlamydomonas reinhardtii has emerged as a model organism for the study of a variety of cellular processes. Posttranslational control via protein phosphorylation plays a key role in signal transduction, regulation of gene expression, and control of metabolism. Thus, analysis of the phosphoproteome of C. reinhardtii can significantly enhance our understanding of various regulatory pathways. In this study, we have grown C. reinhardtii cultures in the presence of an inhibitor of Ser/Thr phosphatases to increase the phosphoprotein pool. Phosphopeptides from these cells were enriched by immobilized metal-ion affinity chromatography and analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS-MS as well as neutral-loss-triggered MS-MS-MS spectra. In this way, we were able to identify 360 phosphopeptides from 328 different phosphoproteins of C. reinhardtii, thus providing new insights into a variety of cellular processes, including metabolic and signaling pathways. Comparative analysis of the phosphoproteome also yielded new functional information on proteins controlled by redox regulation (thioredoxin target proteins) and proteins of the chloroplast 70S ribosome, the centriole, and especially the flagella, for which 32 phosphoproteins were identified. The high yield of phosphoproteins of the latter correlates well with the presence of several flagellar kinases and indicates that phosphorylation/dephosphorylation represents one of the key regulatory mechanisms of eukaryotic cilia. Our data also provide new insights into certain cilium-related mammalian diseases.

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“…Although the most suitable detection method that meets such criteria is still under consideration, there are several candidates, such as 1) fluorescent labeling, [31,32] 2) isotopic labeling, [33,49] 3) chemiluminesent labeling, [69] 4) mass spectrometry, [70] 5) surface plasmon resonance (SPR) spectroscopy, [71][72][73][74][75] 6) anomalous reflections (AR) of the gold surface, [76] 7) quartz-crystal microbalance (QCM) analysis, [77][78][79] 8) fluorescence correlation spectroscopy (FCS), [80][81][82] and 9) electrochemical detection ( Figure 3). It is necessary to label with fluorescent probes for the methods 1 and 8, with radioisotopes for method 2, with an adequate functional group/molecule for method 3, and with electrically active probes for method 9, but no labeling at all is necessary for the other methods (4-7).…”

Section: Detection Methodsmentioning

confidence: 99%

Protein‐Detecting Microarrays: Current Accomplishments and Requirements

2005

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The sequencing of the human genome has been successfully completed and offers the chance of obtaining a large amount of valuable information for understanding complex cellular events simply and rapidly in a single experiment. Interestingly, in addressing these proteomic studies, the importance of protein-detecting microarray technology is increasing. In the coming few years, microarray technology will become a significantly promising and indispensable research/diagnostic tool from just a speculative technology. It is clear that the protein-detecting microarray is supported by three independent but strongly related technologies (surface chemistry, detection methods, and capture agents). Firstly, a variety of surface-modification methodologies are now widely available and offer site-specific immobilization of capture agents onto surfaces in such a way as to keep the native conformation and activity. Secondly, sensitive and parallel detection apparatuses are being developed to provide highly engineered microarray platforms for simultaneous data acquisition. Lastly, in the development of capture agents, antibodies are now probably the most prominent capture agents for analyzing protein abundances. Alternative scaffolds, such as phage-displayed antibody and protein fragments, which provide the advantage of increasing diversity of proteinic capture agents, however, are under development. An approach involving recombinant proteins fused with affinity tag(s) and coupled with a highly engineered surface chemistry will provide simple production protocols and specific orientations of capture agents on the microarray formats. Peptides and other small molecules can be employed in screening highly potent ligands as well as in measuring enzymatic activities. Protein-detecting microarrays supported by the three key technologies should contribute in accelerating diagnostic/biological research and drug discovery.

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An efficient protocol for the identification of protein phosphorylation in a seedless plant, sensitive enough to detect members of signalling cascades

Cited by 47 publications

References 45 publications

Genes, Enzymes, and Regulation of para -Cresol Metabolism in Geobacter metallireducens

Genes, Enzymes, and Regulation of para -Cresol Metabolism in Geobacter metallireducens

Analysis of the Phosphoproteome of Chlamydomonas reinhardtii Provides New Insights into Various Cellular Pathways

Protein‐Detecting Microarrays: Current Accomplishments and Requirements

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