Antonius Koller

Proteomics gives insight into the regulatory function of chloroplast thioredoxins

Thioredoxins are small multifunctional redox active proteins widely if not universally distributed among living organisms. In chloroplasts, two types of thioredoxins (f and m) coexist and play central roles in regulating enzyme activity. Reduction of thioredoxins in chloroplasts is catalyzed by an iron-sulfur disulfide enzyme, ferredoxin-thioredoxin reductase, that receives photosynthetic electrons from ferredoxin, thereby providing a link between light and enzyme activity. Chloroplast thioredoxins function in the regulation of the Calvin cycle and associated processes. However, the relatively small number of known thioredoxin-linked proteins (about 16) raised the possibility that others remain to be identified. To pursue this opportunity, we have mutated thioredoxins f and m, such that the buried cysteine of the active disulfide has been replaced by serine or alanine, and bound them to affinity columns to trap target proteins of chloroplast stroma. The covalently linked proteins were eluted with DTT, separated on gels, and identified by mass spectrometry. This approach led to the identification of 15 potential targets that function in 10 chloroplast processes not known to be thioredoxin linked. Included are proteins that seem to function in plastid-to-nucleus signaling and in a previously unrecognized type of oxidative regulation. Approximately two-thirds of these targets contained conserved cysteines. We also identified 11 previously unknown and 9 confirmed target proteins that are members of pathways known to be regulated by thioredoxin. In contrast to results with individual enzyme assays, specificity for thioredoxin f or m was not observed on affinity chromatography.

Proteomic survey of metabolic pathways in rice

A systematic proteomic analysis of rice (Oryza sativa) leaf, root, and seed tissue using two independent technologies, two-dimensional gel electrophoresis followed by tandem mass spectrometry and multidimensional protein identification technology, allowed the detection and identification of 2,528 unique proteins, which represents the most comprehensive proteome exploration to date. A comparative display of the expression patterns indicated that enzymes involved in central metabolic pathways are present in all tissues, whereas metabolic specialization is reflected in the occurrence of a tissue-specific enzyme complement. For example, tissue-specific and subcellular compartment-specific isoforms of ADP-glucose pyrophosphorylase were detected, thus providing proteomic confirmation of the presence of distinct regulatory mechanisms involved in the biosynthesis and breakdown of separate starch pools in different tissues. In addition, several previously characterized allergenic proteins were identified in the seed sample, indicating the potential of proteomic approaches to survey food samples with regard to the occurrence of allergens.

Protein pathway and complex clustering of correlated mRNA and protein expression analyses in Saccharomyces cerevisiae

The mRNA and protein expression in Saccharomyces cerevisiae cultured in rich or minimal media was analyzed by oligonucleotide arrays and quantitative multidimensional protein identification technology. The overall correlation between mRNA and protein expression was weakly positive with a Spearman rank correlation coefficient of 0.45 for 678 loci. To place the data sets in a proper biological context, a clustering approach based on protein pathways and protein complexes was implemented. Protein expression levels were transcriptionally controlled for not only single loci but for entire protein pathways (e.g., Met, Arg, and Leu biosynthetic pathways). In contrast, the protein expression of loci in several protein complexes (e.g., SPT, COPI, and ribosome) was posttranscriptionally controlled. The coupling of the methods described provided insight into the biology of S. cerevisiae and a clustering strategy by which future studies should be based.

Proteomic characterization of wheat amyloplasts using identification of proteins by tandem mass spectrometry

We describe the initial characterization of the wheat amyloplast proteome, consisting of the identification and classification of 171 proteins. Whole amyloplasts and purified amyloplast membranes were prepared from wheat (Triticum aestivum). Protein extracts were examined by one‐dimensional and two‐dimensional electrophoresis, followed by high performance liquid chromatography‐tandem mass spectrometry of separated proteins. Tandem mass spectrometry data of individual peptides was then searched by SEQUEST, using a database containing known protein sequences from both wheat and other homologous cereal crops. Using this approach we identified 108 proteins from whole amyloplasts and 63 proteins from purified amyloplast membranes. The majority of protein identifications were derived from protein sequences from cereal crops other than wheat, for which relatively little gene sequence data is available. The highest percentage of protein identifications obtained from any individual species was 46% of the total number of proteins identified, using sequence data found in our proprietary rice (Oryza sativa) genome database.

Proteomics uncovers proteins interacting electrostatically with thioredoxin in chloroplasts

The ability of thioredoxin f to form an electrostatic (non-covalent) complex, earlier found with fructose-1,6-bisphosphatase, was extended to include 27 previously unrecognized proteins functional in 11 processes of chloroplasts. The proteins were identified by combining thioredoxin f affinity chromatography with proteomic analysis using tandem mass spectrometry. The results provide evidence that an association with thioredoxin enables the interacting protein to achieve an optimal conformation, so as to facilitate: (i) the transfer of reducing equivalents from the ferredoxin/ferredoxin—thioredoxin reductase complex to a target protein; (ii) in some cases, to enable the channeling of metabolite substrates; (iii) to function as a subunit in the formation of multienzyme complexes.

Peptide Sequencing by Tandem Mass Spectrometry

Publisher Summary Microcolumn reversed-phase HPLC electrospray ionization tandem mass spectrometry (ESI-MS/MS) is a rapid and sensitive technique for the analysis of complex mixtures of peptides. This technique can be used to determine the amino acid sequence of unknown peptides, to verify the structure of proteins, and to determine post-translational modifications. The advantage of low flow rate infusion is that a small volume of sample can be infused over a long time period (5–30 min). This allows time to record a mass spectrum and then to begin acquiring tandem mass spectra of ions observed in the mass spectrum. Low flow rate infusion works quite well for reasonably uncomplicated peptide mixtures. Under low-energy, multiple collision conditions, peptides fragment primarily at the amide bonds, producing sequence-specific fragmentation patterns. When fragment ions are produced the charge can be retained on the N terminus of the fragment of the ion to form ions of type b. If the charge is retained on the C-terminal fragment, the ion is of type y.