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  1. ; ; ; ; ; ( , Rapid Communications in Mass Spectrometry)
    Rationale

    Purification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI).

     Methods

    A protein standard, His‐Ubq, and two recombinant proteins, His‐SHAN and His‐CS, expressed inEscherichia coliwere immobilized on two immobilized metal affinity systems, Cu–nitriloacetic acid (Cu‐NTA) and Ni‐NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96‐well plate form factor, or analyzed directly from immobilized metal affinity‐coated microscope slides by DESI‐MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis.

     Results

    Small proteins (His‐Ubq) and medium proteins (His‐SAHN) could readily be detected from 96‐well plates by direct infusion ESI, or from microscope slides by DESI‐MS after purification on surface from clarifiedE. colicell lysate. Protein oxidation was observed for immobilized proteins on both Cu‐NTA and Ni‐NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His‐SAHN and the methylation product of His‐CS (theobromine to caffeine) were detected.

     Conclusions

    The immobilization, purification, release and detection of His‐tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI‐MS or ambient DESI‐MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.

     
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  2. ; ; ; ; ; ; ; ; ; ( , Proceedings of the National Academy of Sciences)

    Plastid genomes (plastomes) vary enormously in size and gene content among the many lineages of nonphotosynthetic plants, but key lineages remain unexplored. We therefore investigated plastome sequence and expression in the holoparasitic and morphologically bizarre Balanophoraceae. The twoBalanophoraplastomes examined are remarkable, exhibiting features rarely if ever seen before in plastomes or in any other genomes. At 15.5 kb in size and with only 19 genes, they are among the most reduced plastomes known. They have no tRNA genes for protein synthesis, a trait found in only three other plastid lineages, and thusBalanophoraplastids must import all tRNAs needed for translation.Balanophoraplastomes are exceptionally compact, with numerous overlapping genes, highly reduced spacers, loss of allcis-spliced introns, and shrunken protein genes. With A+T contents of 87.8% and 88.4%, theBalanophoragenomes are the most AT-rich genomes known save for a single mitochondrial genome that is merely bloated with AT-rich spacer DNA. Most plastid protein genes inBalanophoraconsist of ≥90% AT, with several between 95% and 98% AT, resulting in the most biased codon usage in any genome described to date. A potential consequence of its radical compositional evolution is the novel genetic code used byBalanophoraplastids, in which TAG has been reassigned from stop to tryptophan. Despite its many exceptional properties, theBalanophoraplastome must be functional because all examined genes are transcribed, its only intron is correctlytrans-spliced, and its protein genes, although highly divergent, are evolving under various degrees of selective constraint.

     
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  3. ; ; ; ( , Proceedings of the National Academy of Sciences)
  4. ; ; ; ; ; ; ; ; ; ; et al ( , Nature)
    Green plants (Viridiplantae) include around 450,000–500,000 species of great diversity and have important roles in terrestrial and aquatic ecosystems. Here, as part of the One Thousand Plant Transcriptomes Initiative, we sequenced the vegetative transcriptomes of 1,124 species that span the diversity of plants in a broad sense (Archaeplastida), including green plants (Viridiplantae), glaucophytes (Glaucophyta) and red algae (Rhodophyta). Our analysis provides a robust phylogenomic framework for examining the evolution of green plants. Most inferred species relationships are well supported across multiple species tree and supermatrix analyses, but discordance among plastid and nuclear gene trees at a few important nodes highlights the complexity of plant genome evolution, including polyploidy, periods of rapid speciation, and extinction. Incomplete sorting of ancestral variation, polyploidization and massive expansions of gene families punctuate the evolutionary history of green plants. Notably, we find that large expansions of gene families preceded the origins of green plants, land plants and vascular plants, whereas whole-genome duplications are inferred to have occurred repeatedly throughout the evolution of flowering plants and ferns. The increasing availability of high-quality plant genome sequences and advances in functional genomics are enabling research on genome evolution across the green tree of life. 
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