Abundant storage protein depletion from tuber proteins using ethanol precipitation method: Suitability to proteomics study

Abstract

High-abundance proteins (HAPs) hamper in-depth proteome study necessitating development of a HAPs depletion method. Here, we report a novel ethanol precipitation method (EPM) for HAPs depletion from total tuber proteins. Ethanol showed a dose-dependent effect on depletion of sporamin from sweet potato and patatin from potato tubers, respectively. The 50% ethanol was an optimal concentration. 2DE analysis of EPM-prepared sweet potato proteins also revealed enrichment of storage proteins (SPs) in ethanol supernatant (ES) resulting in detection of new low-abundance proteins in ethanol pellet (EP), compared to total fraction. The ES fraction showed even higher trypsin inhibitor activity than total proteins, further showing the efficacy of EPM in enrichment of sporamin in ES fraction. Application of this method was demonstrated for comparative proteomics of two sweet potato cultivars (Hwang-geum and Ho-bac) and purification of SP (sporamin) in its native form, as examples. Comparative proteomics identified many cultivar specific protein spots and selected spots were confidently assigned for their protein identity using MALDI-TOF-TOF analysis. Overall, the EPM is simple, reproducible, and economical for depletion of SPs and is suitable for downstream proteomics study. This study opens a door for its potential application to other tuber crops or fruits rich in carbohydrates.

Protein Extraction Methods Shape Much of the Extracted Proteomes

“…Third, complex protein samples can be pre-fractionated to deplete high-abundance proteins, to enhance the detection of “missing” low-abundant proteins (LAPs). For example, the depletion of RuBisCO in leaves (Kim et al, 2013 ; Gupta and Kim, 2015 ) and of storage proteins in seeds (Xiong et al, 2014 ) and tubers (Wu et al, 2012 ; Kim et al, 2015 ; Lee et al, 2015 ; Gupta et al, 2016 ) significantly improved the separation and detection of LAPs.…”

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

“…Ethanol precipitation method was developed to fractionate the sporamin and patatin, major SPs in the sweet potato and potato tubers, respectively ( Lee et al, 2015 ). This method involves extraction of total tuber proteins in aqueous buffer [0.5 M Tris-HCl (pH 8.3), 2% v/v NP-40, 20 mM MgCl 2 and 2% v/v β-mercaptoethanol] followed by incubation of total protein extract with 50% ethanol for 1 h at -20°C.…”

“…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 ).…”

High Abundance Proteins: Proteomer’s Thorns in the Flesh?

“…These depletion based methods utilize different chemicals [12,13], solvents [14] or antibodies against the target proteins [15] to efficiently remove the high-abundance proteins from the total protein samples. Previous reports have shown that depletion of albumin from plasma [15], RuBisCO from leaf [5,12,13] and storage proteins from seed [16,17] and tuber [14] samples lead to the significant increase in the proteome coverage and identification of low-abundance proteins. However, although the precipitation based methods are rapid and costeffective, there is always a risk of loss of some non-target proteins that might precipitate along with the depleted target proteins.…”