Sequential extraction results in improved proteome profiling of medicinal plant Pinellia ternata tubers, which contain large amounts of high-abundance proteins.

Pinellia ternata tuber is one of the well-known Chinese traditional medicines. In order to understand the pharmacological properties of tuber proteins, it is necessary to perform proteome analysis of P. ternata tubers. However, a few high-abundance proteins (HAPs), mainly mannose-binding lectin (agglutinin), exist in aggregates of various sizes in the tubers and seriously interfere with proteome profiling by two-dimensional electrophoresis (2-DE). Therefore, selective depletion of these HAPs is a prerequisite for enhanced proteome analysis of P. ternata tubers. Based on differential protein solubility, we developed a novel protocol involving two sequential extractions for depletion of some HAPs and prefractionation of tuber proteins prior to 2-DE. The first extraction using 10% acetic acid selectively extracted acid-soluble HAPs and the second extraction using the SDS-containing buffer extracted remaining acid-insoluble proteins. After application of the protocol, 2-DE profiles of P. ternata tuber proteins were greatly improved and more protein spots were detected, especially low-abundance proteins. Moreover, the subunit composition of P. ternata lectin was analyzed by electrophoresis. Native lectin consists of two hydrogen-bonded subunits (11 kDa and 25 kDa) and the 11 kDa subunit was a glycoprotein. Subsequently, major HAPs in the tubers were analyzed by mass spectrometry, with nine protein spots being identified as lectin isoforms. The methodology was easy to perform and required no specialized apparatus. It would be useful for proteome analysis of other tuber plants of Araceae

Chloroform-assisted phenol extraction improving proteome profiling of maize embryos through selective depletion of high-abundance storage proteins.

The presence of abundant storage proteins in plant embryos greatly impedes seed proteomics analysis. Vicilin (or globulin-1) is the most abundant storage protein in maize embryo. There is a need to deplete the vicilins from maize embryo extracts for enhanced proteomics analysis. We here reported a chloroform-assisted phenol extraction (CAPE) method for vicilin depletion. By CAPE, maize embryo proteins were first extracted in an aqueous buffer, denatured by chloroform and then subjected to phenol extraction. We found that CAPE can effectively deplete the vicilins from maize embryo extract, allowing the detection of low-abundance proteins that were masked by vicilins in 2-DE gel. The novelty of CAPE is that it selectively depletes abundant storage proteins from embryo extracts of both monocot (maize) and dicot (soybean and pea) seeds, whereas other embryo proteins were not depleted. CAPE can significantly improve proteome profiling of embryos and extends the application of chloroform and phenol extraction in plant proteomics. In addition, the rationale behind CAPE depletion of abundant storage proteins was explored

Determination of Amino Acids and Nucleosides in Medicinal and Edible Hybrid Gastrodia elata and Its Parent Varieties Based on Liquid Chromatography-Tandem Mass Spectrometry

To provide a scientific basis for the follow-up resource utilization and food development of Jinzhai hybrid G. elata, the ultra-high performance liquid chromatography coupled with mass spectrometry was utilized to accurately determine the contents of amino acids and nucleosides in Jinzhai Hongwu hybrid Gastrodia elata Bl. and its parents Yunnan G. elata Bl. f. glauca S. Chow and Jinzhai G. elata Bl. f. elata. Statistical methods were also applied to clarify and comprehensively evaluate the composition and content differences in amino acids and nucleosides among the three varieties of G. elata. C18 column (2.1 mm×100 mm, 1.8 μm) was used with 0.2% formic acid water (A)-acetonitrile (B) as the mobile phase at a flow rate of 0.2 mL/min. The column temperature was 30 ℃ with gradient elution. Moreover, electrospray ion source was measured in multiple reaction monitoring (MRM) mode, which was set as spray voltage at 5500 V; ionization temperature at 550 ℃; ion source gas pressure (Gas) at 241.3 kPa, atomizing gas (GS1) at 379.2 kPa, and auxiliary gas (GS2) at 379.2 kPa. The results revealed that there was a good linear relationship in the range of 0~144.58 μg/mL of the 31 components. The coefficient of determination R2 was greater than 0.999, and the RSD of the precision, stability, and repeatability of the peak area was not greater than 3.0%. The average recovery rate was between 92.69% and 99.5%, while recovery rate RSD ranged from 1.3% to 2.9%. Based on all above, G. elata was rich in amino acids and nucleotides, of which glutamic acid and aspartic acid were the main components. The contents of total amino acids and nucleosides in hybrid G. elata were higher than those of its parents G. elata. And there was a significant difference in the amino acids contents of different varieties (P<0.05). In the evaluation results of PCA and TOPSIS methods, the qualities of S1 and S2 hybrid G. elata were the best. This paper established a new method for evaluating the quality of G. elata and provided a scientific basis for the development and exploitation of Jinzhai hybrid varieties

Proteome Profiling of Maize Pollen Coats Reveals Novel Protein Components

The pollen coat, which covers the exine wall of pollen, is essential for initial sexual contact and thus successful fertilization in flowering plants. Pollen coat proteins (PCPs) not only mediate species specificity but are also needed for pollen–stigma recognition and pollen germination on the stigma. Maize (Zea mays L.) is one of the most common cereal crops in the world. To date, only a few PCPs from maize have been identified and characterized. In the present study, we extracted the pollen coat fraction from maize inbred line B73 with chloroform and purified the PCPs via a phenolbased protocol prior to two-dimensional gel electrophoresis (2-DE). By proteomic analysis, 26 protein spots were successfully identified and classified into 12 unique proteins. The protein composition of the maize pollen coat is distinctly different from those of the dicot plants Arabidopsis and rapeseed. Of the proteins identified in this study, eight (including p r o f i l i ns , caleos i n , Zea m 2 , β- expans i n - 1 0 , exopolygalacturonase, Rho GDP-dissociation inhibitor 1, Ras-related protein Rab-2-A, and putative subtilase) had not previously been observed in the maize pollen coat. Bioinformatic analysis showed that nine of the PCPs were secreted proteins and that most of them were extracellular. These PCPs are potentially involved in pollen germination and tube growth. The current study extracted and identified the pollen coat proteins of maize, one of the most important crops throughout the world. Proteome profiling of the maize pollen coat revealed many novel protein components potentially involved in pollen–stigma interactions and pollen germination. Our results provide basic knowledge and further the functional characterization of PCPs in wind-pollinated species such as maize

Workflow for sequential protein extraction protocol.

<p>Acetone tissue powder of <i>Pinellia ternata</i> tubers was sequentially extracted by 10% acetic acid, followed by SDS-containing buffer.</p

Table_1_Protein Extraction Methods Shape Much of the Extracted Proteomes.DOCX

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Identification of subunit compositions of lectins in <i>P. ternata</i> tubers.

<p><b>A</b>, native gel, total tuber proteins. <b>B</b>, total tuber proteins and purified Bands 1–3 were resolved by SDS-PAGE under reducing conditions (with DTT). <b>C</b>, total tuber proteins and purified Bands 1–3 were resolved by SDS-PAGE under reducing conditions (without DTT).</p

Protein profiles of total proteins of <i>Pinellia ternata</i> tubers separated by SDS-PAGE and 2-DE.

<p>Total protein was extracted from acetone tissue powder of <i>P. ternata</i> tubers with SDS-containing buffer and purified by phenol extraction. Protein loads were 30 μg (<b>A</b>) and 400 μg (<b>B</b>), respectively. Gels were stained with CBB R. HAPs in tuber extract were indicated. Rectangles: indicating the mask effect of HAPS on other protein spots (<b>B</b>).</p

Sequential protein extraction improved the SDS-PAGE profiles of <i>P. ternata</i> tuber proteins.

<p><b>A</b>, equal amounts of proteins (25 μg) from each extract were separated by SDS-PAGE. <b>B</b>, depletion effect of different concentrations of acetic acid. <b>C</b>, detection of glycoproteins in <i>P. ternata</i> tubers. Total tuber proteins were resolved by SDS-PAGE and visualized by CBB (protein load 25 μg) and Alcian stain (protein load 10 μg), respectively. Only the 11 kDa band was stained by Alcian staining, indicating a glycoprotein.</p