Isotopically Dimethyl Labeling-Based Quantitative Proteomic Analysis of Phosphoproteomes of Soybean Cultivars.

Isotopically dimethyl labeling was applied in a quantitative post-translational modification (PTM) proteomic study of phosphoproteomic changes in the drought responses of two contrasting soybean cultivars. A total of 9457 phosphopeptides were identified subsequently, corresponding to 4571 phosphoprotein groups and 3889 leading phosphoproteins, which contained nine kinase families consisting of 279 kinases. These phosphoproteins contained a total of 8087 phosphosites, 6106 of which were newly identified and constituted 54% of the current soybean phosphosite repository. These phosphosites were converted into the highly conserved kinase docking sites by bioinformatics analysis, which predicted six kinase families that matched with those newly found nine kinase families. The overly post-translationally modified proteins (OPP) occupies 2.1% of these leading phosphoproteins. Most of these OPPs are photoreceptors, mRNA-, histone-, and phospholipid-binding proteins, as well as protein kinase/phosphatases. The subgroup population distribution of phosphoproteins over the number of phosphosites of phosphoproteins follows the exponential decay law, Y = 4.13e-0.098X - 0.04. Out of 218 significantly regulated unique phosphopeptide groups, 188 phosphoproteins were regulated by the drought-tolerant cultivar under the water loss condition. These significantly regulated phosphoproteins (SRP) are mainly enriched in the biological functions of water transport and deprivation, methionine metabolic processes, photosynthesis/light reaction, and response to cadmium ion, osmotic stress, and ABA response. Seventeen and 15 SRPs are protein kinases/phosphatases and transcription factors, respectively. Bioinformatics analysis again revealed that three members of the calcium dependent protein kinase family (CAMK family), GmSRK2I, GmCIPK25, and GmAKINß1 kinases, constitute a phosphor-relay-mediated signal transduction network, regulating ion channel activities and many nuclear events in this drought-tolerant cultivar, which presumably contributes to the development of the soybean drought tolerance under water deprivation process.

SILIA-Based 4C Quantitative PTM Proteomics.

To absolutely and relatively quantitate the alteration of a posttranslationally modified (PTM) proteome in response to a specific internal or external signal, a 15N-stable isotope labeling in Arabidopsis (SILIA) protocol has been integrated into the 4C quantitative PTM proteomics, named as SILIA-based 4C quantitative PTM proteomics (S4Quap). The isotope metabolic labeling produces both forward (F) and reciprocal (R) mixings of either 14N/15N-coded tissues or the 14N/15N-coded total cellular proteins. Plant protein is isolated using a urea-based extraction buffer (UEB). The presence of 8 M urea, 2% polyvinylpolypyrrolidone (PVPP), and 5 mM ascorbic acid allows to instantly denature protein, remove the phenolic compounds, and curb the oxidation by free radicals once plant cells are broken. The total cellular proteins are routinely processed into peptides by trypsin. The PTM peptide yield of affinity enrichment and preparation is 0.1-0.2% in general. Ion exchange chromatographic fractionation prepares the PTM peptides for LC-MS/MS analysis. The collected mass spectrograms are subjected to a target-decoy sequence analysis using various search engines. The computational programs are subsequently applied to analyze the ratios of the extracted ion chromatogram (XIC) of the 14N/15N isotope-coded PTM peptide ions and to perform the statistical evaluation of the quantitation results. The Student t-test values of ratios of quantifiable 14N/15N-coded PTM peptides are normally corrected using a Benjamini-Hochberg (BH) multiple hypothesis test to select the significantly regulated PTM peptide groups (BH-FDR < 5%). Consequently, the highly selected prospect candidate(s) of PTM proteins are confirmed and validated using biochemical, molecular, cellular, and transgenic plant analysis.

Isoorientin derived from Gentiana veitchiorum Hemsl. flowers inhibits melanogenesis by down-regulating MITF-induced tyrosinase expression.

BACKGROUND: Gentiana is a genus of flowering plants in Gentianaceae family, which comprises of 1,600 species. The roots of few species of Gentiana, also known as Long Dan Cao in Chinese, are traditionally used in herbal remedies for a wide variety of liver-associated diseases. The medicinal part of Gentiana is root; however, the trumpet-shaped flowers are seldom being used. PURPOSE: We investigated the anti-melanogenesis effect of water extract of Gentiana veitchiorum Hemsl. flowers, and isoorientin was identified to be the active compound. STUDY DESIGN: We tested the anti-melanogenesis effects of extracts deriving from different parts of G. veitchiorum, followed by identification of active ingredients within the extracts. The mechanism of inhibitory effect on melanogenesis, triggered by isoorientin, was elucidated by in vitro analyses. METHODS: HPLC was applied to identify the components in water extracts from different parts of G. veitchiorum. The cytotoxicity of extracts and pure compounds in cultured B16F10 murine melanoma cells was determined by MTT and trypan blue assays. Melanin assay, tyrosinase assay, RT-PCR, luciferase assay and western blot were used to analyze the effect of isoorientin in melanin content, tyrosinase activity, as well as the expressions of those related genes and proteins. RESULTS: We identified an inhibitory effect on melanogenesis from water extract of G. veitchiorum flowers in B16F10 cells. Isoorientin, a major flavone in the extract, was identified to be an active ingredient causing reduction in melanin content in a dose-dependent manner. Such reduction was suggested to be a result of suppressed expression of tyrosinase (TYR), tyrosinase related protein-1 (TRP1) and DOPA-chrome tautomerase (DCT). Isoorientin also suppressed the expression of microphthalmia- associated transcription factor (MITF) through the phosphorylation of cAMP response element-binding protein (CREB). CONCLUSION: These findings indicate that isoorientin derived from G. veitchiorum flowers may be a potential skin lightening agent for the treatment of skin pigmentary disorders.