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Wolfram Weckwerth

Publications and source records attributed to Wolfram Weckwerth.

At least 19 recordsLinked to original sources
PubMed2007

Integrative profiling of metabolites and proteins: improving pattern recognition and biomarker selection for systems level approaches.

Methods such as mRNA expression profiling have provided a vast amount of genomic and transcriptomic information about plants and other organisms. However, there is explicit indication that considerable metabolic control is executed on the metabolite and on the protein level including protein modifications, thereby constituting the phenotypic plasticity. Consequently, the analysis of the molecular phenotype demands the step toward mass spectrometry (MS)-based postgenomic techniques such as metabolomics and proteomics. This chapter describes a straightforward protocol for simultaneously extracting metabolites and proteins from the same biological sample in preparation for MS analysis. Furthermore, protocols for profiling polar metabolites using gas chromatography time-of-flight MS and for shotgun proteomics using liquid chromatography-MS are discussed. A practical course is laid out that outlines all the basic steps, from harvesting to data analysis. These steps enable the correlative study of metabolite and protein dynamics with minimal technical variation. Biological variability of independent samples is exploited for variance analysis and pattern recognition.

Arabidopsis↗
PubMed2007

A gentle guide to the analysis of metabolomic data.

Modern molecular biology crucially relies on computational tools to handle and interpret the large amounts of data that are generated by high-throughput measurements. To this end, much effort is dedicated to devise novel sophisticated methods that allow one to integrate, evaluate, and analyze biological data. However, prior to an application of specifically designed methods, simple and well-known statistical approaches often provide a more appropriate starting point for further analysis. This chapter seeks to describe several well-established approaches to data analysis, including various clustering techniques, discriminant function analysis, principal component analysis, multidimensional scaling, and classification trees. The chapter is accompanied by a webpage, describing the application of all algorithms in a ready-to-use format.

Arabidopsis↗
PubMed2006

Relative and absolute quantitative shotgun proteomics: targeting low-abundance proteins in Arabidopsis thaliana.

The plant system is a highly dynamic structure on all molecular levels, transcripts, proteins, and metabolites. Thus, protein analysis has to cope with a highly dynamic range of concentrations. A severe problem is the detection of low-abundance proteins in the presence of housekeeping proteins. Basically three approaches are facilitated to measure protein abundance in a comprehensive manner: 2DE and one- or multi-dimensional shotgun proteomics, with or without stable-isotope labelling. These comparative techniques allow for the identification of altered protein levels compared with a reference state. However, they are limited to the analysis of medium/high-abundance proteins. Using stable-isotope dilution techniques it is possible to target the quantitative analysis to low-abundance proteins and to measure absolute concentrations of proteins. Based on multi-dimensional non-gel shotgun proteomics in Arabidopis thaliana, a list of tryptic peptides comprising >1000 proteins was generated. A strategy for quantitative plant proteomics is proposed using this master-list for selecting signature peptides of proteins. To prove the concept, a liquid chromatography-high-resolution triple quadrupole multiple reaction monitoring-mass spectrometry technique is described to determine the absolute amount of a low-abundance sucrose synthase isoform out of an ultra-complex A. thaliana protein extract.

Arabidopsis↗
PubMed2005

Metabolomics: from pattern recognition to biological interpretation.

Metabolomics is a technology that aims to identify and quantify the metabolome -- the dynamic set of all small molecules present in an organism or a biological sample. In this sense, the technique is distinct from metabolic profiling, which looks for target compounds and their biochemical transformation. The combination of both approaches is an emerging technique for the characterization of biological samples and for drug treatment. Metabolomics has proven to be very rapid and superior to any other post-genomics technology for pattern-recognition analyses of biological samples. Changing steady state concentrations and fluctuations of metabolites that occur within milliseconds are a result of biochemical processes such as signalling cascades: metabolomic techniques are instrumental in measuring these changes rapidly and sensitively. Metabolite data can be complemented by protein, transcript and external (environmental) data, thereby leading to the identification of multiple physiological biomarkers embedded in correlative molecular networks that are not approachable with targeted studies.

Biomarkers↗
PubMed2005

Combining metal oxide affinity chromatography (MOAC) and selective mass spectrometry for robust identification of in vivo protein phosphorylation sites.

BACKGROUND: Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the Arabidopsis proteome, however, only a few studies look systematically for in vivo protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy. RESULTS: Here, a strategy is presented that combines enrichment of phosphoproteins using a technique termed metaloxide affinity chromatography (MOAC) and selective ion trap mass spectrometry. The complete approach involves (i) enrichment of proteins with low phosphorylation stoichiometry out of complex mixtures using MOAC, (ii) gel separation and detection of phosphorylation using specific fluorescence staining (confirmation of enrichment), (iii) identification of phosphoprotein candidates out of the SDS-PAGE using liquid chromatography coupled to mass spectrometry, and (iv) identification of phosphorylation sites of these enriched proteins using automatic detection of H3PO4 neutral loss peaks and data-dependent MS3-fragmentation of the corresponding MS2-fragment. The utility of this approach is demonstrated by the identification of phosphorylation sites in Arabidopsis thaliana seed proteins. Regulatory importance of the identified sites is indicated by conservation of the detected sites in gene families such as ribosomal proteins and sterol dehydrogenases. To demonstrate further the wide applicability of MOAC, phosphoproteins were enriched from Chlamydomonas reinhardtii cell cultures. CONCLUSION: A novel phosphoprotein enrichment procedure MOAC was applied to seed proteins of A. thaliana and to proteins extracted from C. reinhardtii. Thus, the method can easily be adapted to suit the sample of interest since it is inexpensive and the components needed are widely available. Reproducibility of the approach was tested by monitoring phosphorylation sites on specific proteins from seeds and C. reinhardtii in duplicate experiments. The whole process is proposed as a strategy adaptable to other plant tissues providing high confidence in the identification of phosphoproteins and their corresponding phosphorylation sites.

Journal Article↗
PubMed2005

Differential multisite phosphorylation of the trehalose-6-phosphate synthase gene family in Arabidopsis thaliana: a mass spectrometry-based process for multiparallel peptide library phosphorylation analysis.

Multisite protein phosphorylation plays a fundamental role in metabolic regulation. To detect and quantify in vitro kinase phosphorylation activities, we developed a highly selective LC-MS/MS-based method using high resolution multiple reaction monitoring on a triple quadrupole mass spectrometer. This method eliminates the need for stable isotope labeling and enables multiparallel kinase target assays. Using these assays, we made the first observation of in vitro phosphorylation of different trehalose-6-phosphate synthase (TPS) isozymes. TPSs possess putative Ca2+-independent, sucrose non-fermenting 1-related protein kinase 1 (SnRK1) phosphorylation sites. Sixteen synthetic peptides from six different Arabidopsis thaliana TPS isozymes containing the SnRK1 consensus recognition motif were phosphorylated simultaneously in vitro, and their phosphorylation dynamics were determined. We achieved absolute quantification of TPS peptide phosphorylation by tuning the mass spectrometer to the corresponding synthetic standard phosphopeptides. The selectivity of the mass spectrometer in the multiple reaction monitoring mode compensates for the low ionization efficiency of phosphopeptides in the presence of a complex matrix. Results are in close agreement with recent in vivo studies of TPS phosphorylation and regulation and reveal significant differences in the phosphorylation levels of different TPS members within the TPS gene family ranging over 3 orders of magnitude. Substituting EGTA for CaCl2 in the reaction mixture reduced the formation of some of the phospho-TPS peptides drastically, indicating that Ca2+-dependent kinases are active in the presence of Ca2+-independent SnRKs. This agrees with the proposed overlap of the consensus motifs of these kinases and enables delineation between Ca2+-independent and Ca2+-dependent phosphorylation. Results demonstrate that multiparallel kinase target assays are sensitive enough to provide evidence for differential multisite phosphorylation of homologous TPS proteins and their highly conserved putative phosphorylation sites.

Amino Acid Sequence↗
PubMed2005

Species-specific analysis of protein sequence motifs using mutual information.

BACKGROUND: Protein sequence motifs are by definition short fragments of conserved amino acids, often associated with a specific function. Accordingly protein sequence profiles derived from multiple sequence alignments provide an alternative description of functional motifs characterizing families of related sequences. Such profiles conveniently reflect functional necessities by pointing out proximity at conserved sequence positions as well as depicting distances at variable positions. Discovering significant conservation characteristics within the variable positions of profiles mirrors group-specific and, in particular, evolutionary features of the underlying sequences. RESULTS: We describe the tool PROfile analysis based on Mutual Information (PROMI) that enables comparative analysis of user-classified protein sequences. PROMI is implemented as a web service using Perl and R as well as other publicly available packages and tools on the server-side. On the client-side platform-independence is achieved by generally applied internet delivery standards. As one possible application analysis of the zinc finger C2H2-type protein domain is introduced to illustrate the functionality of the tool. CONCLUSION: The web service PROMI should assist researchers to detect evolutionary correlations in protein profiles of defined biological sequences. It is available at http://promi.mpimp-golm.mpg.de where additional documentation can be found.

Amino Acid Motifs↗
PubMed2005

D-GLYCERATE 3-KINASE, the last unknown enzyme in the photorespiratory cycle in Arabidopsis, belongs to a novel kinase family.

D-GLYCERATE 3-KINASE (GLYK; EC 2.7.1.31) catalyzes the concluding reaction of the photorespiratory C2 cycle, an indispensable ancillary metabolic pathway to the photosynthetic C3 cycle that enables land plants to grow in an oxygen-containing atmosphere. Except for GLYK, all other enzymes that contribute to the C2 cycle are known by their primary structures, and the encoding genes have been identified. We have purified and partially sequenced this yet missing enzyme from Arabidopsis thaliana and identified it as a putative kinase-annotated single-copy gene At1g80380. The exclusive catalytic properties of the gene product were confirmed after heterologous expression in Escherichia coli. Arabidopsis T-DNA insertional knockout mutants show no GLYK activity and are not viable in normal air; however, they grow under elevated CO2, providing direct evidence of the obligatory nature of the ultimate step of the C2 cycle. The newly identified GLYK is both structurally and phylogenetically distinct from known glycerate kinases from bacteria and animals. Orthologous enzymes are present in other plants, fungi, and some cyanobacteria. The metabolic context of GLYK activity in fungi and cyanobacteria remains to be investigated.

Amino Acid Sequence↗
PubMed2005

Enrichment of phosphorylated proteins and peptides from complex mixtures using metal oxide/hydroxide affinity chromatography (MOAC).

A novel method termed metal oxide affinity chromatography (MOAC) of enriching for phosphorylated proteins and peptides based on the affinity of the phosphate group for Al(OH)(3) is presented here. When compared to commercial phosphoprotein-enrichment kits, this method is more selective, more cost effective and easily applicable to method optimization. The use of glutamic and aspartic acid in the loading buffer significantly enhances selectivity. Standard protein mixtures and complex Arabidopsis thaliana leaf protein extracts were tested for efficacy of enrichment. The method can be applied to proteins extracted using either mild or denaturing conditions. The same Al(OH)(3) material is suitable for the enrichment of phosphopeptides out of a tryptic digest of alpha-casein. Peptide phosphorylation was revealed by beta-elimination of phosphate groups. Enrichment and in vivo phosphorylation of A. thaliana leaf proteins were confirmed with Pro-Q diamond stain. Several of the phosphoprotein candidates that were identified by MS are known to be phosphorylated in vivo in other plant species.

Aluminum Hydroxide↗
PubMed2005

An integrated strategy for identification and relative quantification of site-specific protein phosphorylation using liquid chromatography coupled to MS2/MS3.

Reversible and differential multisite protein phosphorylation is an important mechanism controlling the activity of cellular proteins. Here we describe a robust and highly selective approach for the identification and relative quantification of site-specific phosphorylation events. This integrated strategy has three major parts: visualisation of phosphorylated proteins using fluorescently stained polyacrylamide gels, determination of the phosphorylation site(s) using automatic MS3 triggered by the loss of phosphoric acid, and relative quantification of phosphorylation by integrating MS2- and MS3-extracted ion traces using a fast-scanning, linear ion trap mass spectrometer. As a test case, recombinant sucrose-phosphate synthase (SPS) from Arabidopsis thaliana (At5g1110) was used for identification and quantification of site-specific phosphorylation. The identified phosphorylation site of the actively expressed protein coincides with the major regulatory in vivo phosphorylation site in spinach SPS. Site-specific differential in vitro phosphorylation of native protein was demonstrated after incubation of the recombinant protein with cold-adapted plant leaf extracts from A. thaliana, suggesting regulatory phosphorylation events of this key enzyme under stress response.

Acclimatization↗
PubMed2004

GMD@CSB.DB: the Golm Metabolome Database.

UNLABELLED: Metabolomics, in particular gas chromatography-mass spectrometry (GC-MS) based metabolite profiling of biological extracts, is rapidly becoming one of the cornerstones of functional genomics and systems biology. Metabolite profiling has profound applications in discovering the mode of action of drugs or herbicides, and in unravelling the effect of altered gene expression on metabolism and organism performance in biotechnological applications. As such the technology needs to be available to many laboratories. For this, an open exchange of information is required, like that already achieved for transcript and protein data. One of the key-steps in metabolite profiling is the unambiguous identification of metabolites in highly complex metabolite preparations from biological samples. Collections of mass spectra, which comprise frequently observed metabolites of either known or unknown exact chemical structure, represent the most effective means to pool the identification efforts currently performed in many laboratories around the world. Here we present GMD, The Golm Metabolome Database, an open access metabolome database, which should enable these processes. GMD provides public access to custom mass spectral libraries, metabolite profiling experiments as well as additional information and tools, e.g. with regard to methods, spectral information or compounds. The main goal will be the representation of an exchange platform for experimental research activities and bioinformatics to develop and improve metabolomics by multidisciplinary cooperation. AVAILABILITY: http://csbdb.mpimp-golm.mpg.de/gmd.html CONTACT: Steinhauser@mpimp-golm.mpg.de SUPPLEMENTARY INFORMATION: http://csbdb.mpimp-golm.mpg.de/

Database Management Systems↗
PubMed2004

Metabolite profiling in plant biology: platforms and destinations.

Optimal use of genome sequences and gene-expression resources requires powerful phenotyping platforms, including those for systematic analysis of metabolite composition. The most used technologies for metabolite profiling, including mass spectral, nuclear magnetic resonance and enzyme-based approaches, have various advantages and disadvantages, and problems can arise with reliability and the interpretation of the huge datasets produced. These techniques will be useful for answering important biological questions in the future.

Biology↗
PubMed2004

Differential metabolic networks unravel the effects of silent plant phenotypes.

Current efforts aim to functionally characterize each gene in model plants. Frequently, however, no morphological or biochemical phenotype can be ascribed for antisense or knock-out plant genotypes. This is especially the case when gene suppression or knockout is targeted to isoenzymes or gene families. Consequently, pleiotropic effects and gene redundancy are responsible for phenotype resistance. Here, techniques are presented to detect unexpected pleiotropic changes in such instances despite very subtle changes in overall metabolism. The method consists of the relative quantitation of >1,000 compounds by GC/time-of-flight MS, followed by classical statistics and multivariate clustering. Complementary to these tools, metabolic networks are constructed from pair-wise analysis of linear metabolic correlations. The topology of such networks reflects the underlying regulatory pathway structure. A differential analysis of network connectivity was applied for a silent potato plant line suppressed in expression of sucrose synthase isoform II. Metabolic alterations could be assigned to carbohydrate and amino acid metabolism even if no difference in average metabolite levels was found.

Analysis of Variance↗
PubMed2004

Process for the integrated extraction, identification and quantification of metabolites, proteins and RNA to reveal their co-regulation in biochemical networks.

A novel extraction protocol is described with which metabolites, proteins and RNA are sequentially extracted from the same sample, thereby providing a convenient procedure for the analysis of replicates as well as exploiting the inherent biological variation of independent samples for multivariate data analysis. A detection of 652 metabolites, 297 proteins and clear RNA bands in a single Arabidopsis thaliana leaf sample was validated by analysis with gas chromatography coupled to a time of flight mass spectrometer for metabolites, two-dimensional liquid chromatography coupled to mass spectrometry for proteins, and Northern blot analysis for RNA. A subset of the most abundant proteins and metabolites from replicate analysis of different Arabidopsis accessions was merged to form an integrative dataset allowing both classification of different genotypes and the unbiased analysis of the hierarchical organization of proteins and metabolites within a real biochemical network.

Arabidopsis↗
PubMed2004

Linking protein fractionation with multidimensional monolithic reversed-phase peptide chromatography/mass spectrometry enhances protein identification from complex mixtures even in the presence of abundant proteins.

Recently, multidimensional shotgun proteomics has proven to be an alternative technology able to identify hundreds of proteins from single samples. Two major limitations of the technology are the presence of high abundance proteins (e.g. RUBISCO in plant leaf tissue) and the enormous number of co-eluting peptides that overstrain the loading and resolving capacity of conventional particle-packed columns as well as the capacity of electrospray ionisation due to ion suppression. Here, the coupling of fast performance liquid chromatography (FPLC) pre-fractionation of an Arabidopsis leaf protein extract and subsequent two-dimensional liquid chromatography/mass spectrometry with improved resolution using a monolithic silica C18 capillary column allowed the identification of 1032 unique proteins in a single 4 mg total protein plant leaf tissue sample. The reassignment of peptide IDs to distinct FPLC protein fractions enhances the identification procedure, especially in the case of present protein isoforms. The proposed strategy is useful to detect proteins otherwise not seen in conventional multidimensional chromatography/mass spectrometry approaches.

Amino Acid Sequence↗
PubMed2004

Cell-specific protein profiling in Arabidopsis thaliana trichomes: identification of trichome-located proteins involved in sulfur metabolism and detoxification.

Metabolite, protein, and transcript analysis at the cellular level gives unparalleled insight into the complex roles tissues play in the plant system. However, while capillary electrophoresis and PCR amplification strategies make the profiling of metabolites and transcripts in specific cell types possible, the profiling of proteins in small samples represents a bottleneck. Here for the first time protein profiling has been achieved in a specific plant cell type: The application of specific cell sampling and shotgun peptide sequencing (nano LC/MS/MS) resulted in the identification of 63 unique proteins from pooled Arabidopsis trichome cells. A complete S-adenosylmethionine pathway cluster, two S-adenosylmethionine synthase isoforms, a glutathione S-conjugate translocator and other proteins involved in sulfur metabolism and detoxification are shown to be present in these cells, in agreement with previous work done at the level of trichome transcript analysis. The technology described here brings the simultaneous identification and localization of physiologically relevant cellular proteins within reach.

Arabidopsis↗
PubMed2003

Scoring and identifying organism-specific functional patterns and putative phosphorylation sites in protein sequences using mutual information.

The co-variance of amino acid positions within a multiple alignment of 294 protein kinases from mammals, plants, and bacteria was studied. Applying mutual information (MI), characteristic amino acid sites have been identified markedly discriminating the different organisms. The relation of surface accessibility of these sites in the 3D structure of a kinase and their MI content is studied. We extended the method to score a predicted phosphorylation site of this highly conserved catalytic protein kinase region. Based on this score mammalian and plant protein kinases were grouped together apart from the bacterial kinases. Thus, the presented method allows us to analyse putative phosphorylation sites in the context of their organism-specific origin.

Amino Acid Sequence↗