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Publications (3)10.43 Total impact

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    ABSTRACT: Bottom-up, or shotgun, proteomics typically relies heavily on trypsin for its ease-of-use and reproducibility. Nonetheless, the need for greater sequencing depth and coverage has led researchers to develop complementary digestion approaches using other proteases. One such protease, endo-Lys-N [EC 3.4.24.20] (Lys-N), has been explored in considerable detail for its use in proteomics. Some of the advantages of Lys-N include the ability to perform digestions in strongly-denaturing conditions and its ability to produce peptides that yield ETD spectra which are generally less complex than their corresponding LysC or tryptic peptides. This attribute is particularly advantageous for de novo sequencing efforts by MS. Despite these advantages, the high cost of commercially-available Lys-N poses a barrier to its wider use within the research community. Here, we describe a novel protocol for a rapid purification of Lys-N from store-bought mushrooms which exploits both its thermal stability and high affinity for carboxymethyl cellulose. The resulting preparation exhibits comparable performance to a commercially available enzyme, as evidenced by LC-MS/MS analysis. The isolation can be completed in a few hours, and yields active enzyme at a fraction of the commercial cost.
    Current Proteomics 01/2013; 10(4). · 0.83 Impact Factor
  • Prashanthi Vallabhaneni, W Keith Ray, Brenda S J Winkel, Richard F Helm
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    ABSTRACT: Developments in mass spectrometry-based technologies are offering insights into the complexity and dynamic nature of plant metabolism. However, the ability to generate reliable metabolic profiles at high spatial resolution is still limited by the need of most technologies for large sample sizes or time-intensive extraction and detection methods. Here we describe the use of flow injection electrospray mass spectrometry for the rapid identification and semi-quantitative analysis of flavonol glycosides in individual root tips. This method uncovered spatial and temporal differences in metabolic profiles that were masked in analyses of whole roots or seedlings, while showing that individual biological replicates can be extremely consistent.
    Phytochemistry 11/2011; 73(1):114-8. · 3.05 Impact Factor
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    ABSTRACT: Auxin and ethylene are key regulators of plant growth and development, and thus the transcriptional networks that mediate responses to these hormones have been the subject of intense research. This study dissected the hormonal cross talk regulating the synthesis of flavonols and examined their impact on root growth and development. We analyzed the effects of auxin and an ethylene precursor on roots of wild-type and hormone-insensitive Arabidopsis (Arabidopsis thaliana) mutants at the transcript, protein, and metabolite levels at high spatial and temporal resolution. Indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) differentially increased flavonol pathway transcripts and flavonol accumulation, altering the relative abundance of quercetin and kaempferol. The IAA, but not ACC, response is lost in the transport inhibitor response1 (tir1) auxin receptor mutant, while ACC responses, but not IAA responses, are lost in ethylene insensitive2 (ein2) and ethylene resistant1 (etr1) ethylene signaling mutants. A kinetic analysis identified increases in transcripts encoding the transcriptional regulators MYB12, Transparent Testa Glabra1, and Production of Anthocyanin Pigment after hormone treatments, which preceded increases in transcripts encoding flavonoid biosynthetic enzymes. In addition, myb12 mutants were insensitive to the effects of auxin and ethylene on flavonol metabolism. The equivalent phenotypes for transparent testa4 (tt4), which makes no flavonols, and tt7, which makes kaempferol but not quercetin, showed that quercetin derivatives are the inhibitors of basipetal root auxin transport, gravitropism, and elongation growth. Collectively, these experiments demonstrate that auxin and ethylene regulate flavonol biosynthesis through distinct signaling networks involving TIR1 and EIN2/ETR1, respectively, both of which converge on MYB12. This study also provides new evidence that quercetin is the flavonol that modulates basipetal auxin transport.
    Plant physiology 03/2011; 156(1):144-64. · 6.56 Impact Factor