Abstract
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.
Highlights
Pinellia ternata, a member of the Araceae family, is a perennial monocot herb native to Asia and grows as an invasive weed in parts of North America [1]
We found that a few highabundance proteins (HAPs) existed in large amounts in the tubers and seriously interfered with proteome profiling of P. ternata tubers
As the first step in our proteome analysis strategy, we developed a sequential extraction protocol to deplete lectin and prefractionate proteins of P. ternata tubers
Summary
A member of the Araceae family, is a perennial monocot herb native to Asia and grows as an invasive weed in parts of North America [1]. It is expected that proteome analysis of P. ternata tubers will provide much information on protein constituents and help to elucidate the pharmacological properties of tuber proteins of interest. Its gene constitutively expresses in various tissues including tuber, leaf, stem and inflorescence [6] Another characterized P. ternata lectin is a 6 kDa glycoprotein, with contents from 5.75 to 8.30% in the tubers [7]. To better understand the regulation mechanism of lectin biosynthesis, it is useful to perform proteome analysis of P. ternata tubers. As the first step in our proteome analysis strategy, we developed a sequential extraction protocol to deplete lectin and prefractionate proteins of P. ternata tubers. The protocol allowed for the enhanced P. ternata tubers proteome analysis and detection of more protein spots, especially LAPs. The recovery and solubilization of sequentially extracted proteins were optimized. The methodology would be useful for the proteomic analysis of other tuber species of Araceae
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