Chloroform-Assisted Phenol Extraction Improving Proteome Profiling of Maize Embryos through Selective Depletion of High-Abundance Storage Proteins

Abstract

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.

Accumulation Profiles of Embryonic Salt-Soluble Proteins in Maize Hybrids and Parental Lines Indicate Matroclinous Inheritance: A Proteomic Analysis

“…The immunoblot analysis ascertained anti-GLB1 was highly specific for 60–70 kDa proteins with a strong reactivity. 2-DE blot analysis also confirmed the specificity of the antibody for the detection of GLB1 proteoforms (Figure 3 , Figure S2 ), which were identified by MS/MS in our previous study (Xiong et al, 2014 ).…”

“…GLB1 was purified as described (Xiong et al, 2014 ) with some modifications. The dried powder of embryo tissues (0.2 g) was homogenized in a cold mortar in 1.0 ml of buffer containing 0.25 M Tris-HCl (pH 7.5), 1% SDS, 14 mM DTT, 5 mM EDTA, and 2 mM PMSF.…”

Expect the Unexpected Enrichment of “Hidden Proteome” of Seeds and Tubers by Depletion of Storage Proteins

“…Chloroform-assisted Phenol Extraction (CAPE) method was developed to deplete the vicilins, major SPs in maize embryos ( Xiong et al, 2014 ). This method involves extraction of seed proteins first in aqueous buffer [0.25 M Tris-HCl (pH 7.5), 1% SDS, 14 mM DTT and a cocktail of protease inhibitors] followed by denaturation of proteins by chloroform [1:1 (v:v) of extract:chloroform, shaking for 10 min] and finally extraction of proteins using the phenol-methanolic ammonium acetate precipitation method.…”

“…MS/MS identification of the 17 depleted spots, confirmed those to be the vicilins, further indicated the efficacy of the CAPE in selective depletion of SPs from maize seeds. Moreover, the application of this method was extended in soybean where the depletion of glycinin and β-conglycinin subunits was shown following this protocol ( Xiong et al, 2014 ).…”

“…A major function of these SPs is to act as a source of carbon and nitrogen during the seed and tuber germination, however, some of these proteins also possess enzymatic activities and exhibit beneficial health effects ( Shewry, 1995 ) ( Agrawal and Rakwal, 2012 ). Glycinin and β-conglycinin in soybean seeds ( Natarajan, 2014 ), vicilins in maize seeds ( Xiong et al, 2014 ), sporamin in sweet potato tubers ( Lee et al, 2015 ), patatin in potato tubers ( Lee et al, 2015 ), and dioscorin in yam tubers ( Xue et al, 2015 ), are some of the common examples of SPs. The seed SPs have been classified into five classes on the basis of their solubility in water (albumins), dilute saline solutions (globulins), alcohol-water mixtures (prolamins), and dilute alkali/acid (glutelins) ( Shewry, 1995 ) and ( Miernyk and Johnston, 2012 ).…”