A protein extraction method for low protein concentration solutions compatible with the proteomic analysis of rubber particles

Abstract

The extraction of high-purity proteins from the washing solution (WS) of rubber particles (also termed latex-producing organelles) from laticifer cells in rubber tree for proteomic analysis is challenging due to the low concentration of proteins in the WS. Recent studies have revealed that proteins in the WS might play crucial roles in natural rubber biosynthesis. To further examine the involvement of these proteins in natural rubber biosynthesis, we designed an efficiency method to extract high-purity WS proteins. We improved our current borax and phenol-based method by adding reextraction steps with phenol (REP) to improve the yield from low protein concentration samples. With this new method, we extracted WS proteins that were suitable for proteomics. Indeed, compared to the original borax and phenol-based method, the REP method improved both the quality and quantity of isolated proteins. By repeatedly extracting from low protein concentration solutions using the same small amount of phenol, the REP method yielded enough protein of sufficiently high-quality from starting samples containing less than 0.02 mg of proteins per milliliter. This method was successfully applied to extract the rubber particle proteins from the WS of natural rubber latex samples. The REP-extracted WS proteins were resolved by 2DE, and 28 proteins were positively identified by MS. This method has the potential to become widely used for the extraction of proteins from low protein concentration solutions for proteomic analysis.

Identification and Characterization of Glycoproteins and Their Responsive Patterns upon Ethylene Stimulation in the Rubber Latex

“…It was identified by mass spectrometry from the latex of Opium poppy [ 49 ] and the milky sap of C. majus [ 45 ]. Phosphate uridylyltransferase was determined as an ERGP in rubber latex in this study ( Table S2 ), and it was also identified from the washed solutions of rubber particles [ 53 ] and lettuce latex in a proteomics study [ 47 ].…”

Physiological and Proteomic Analyses of Molybdenum- and Ethylene-Responsive Mechanisms in Rubber Latex

“…Recently, many proteins had been identified by proteomics-based technologies from different Hevea latex components, such as total latex ( D’Amato et al, 2010 ; Li et al, 2011 ; Wang et al, 2015 ; Dai et al, 2016 ; Habib et al, 2017 ), rubber particles ( Rojruthai et al, 2010 ; Xiang et al, 2012 ; Dai et al, 2013 , 2016 ; Wang et al, 2016 ; Yamashita et al, 2016 ), C-serum ( Wagner et al, 2007 ; Wang et al, 2010 ; Havanapan et al, 2016 ), and lutoids ( Wang et al, 2010 , 2013 ; Habib et al, 2017 ). Proteins involved in mevalonate (MVA) pathway are crucial for NRB ( Wang et al, 2015 ).…”

Development of an Efficient Protein Extraction Method Compatible with LC-MS/MS for Proteome Mapping in Two Australian Seagrasses Zostera muelleri and Posidonia australis

“…Incorporation of ascorbic acid, borax, PVPP, and β-mercaptoethanol into the protein extraction buffer have been previously suggested to extract storage proteins from the recalcitrant tissues of olive leaf (Garcia et al, 2000 ), from tropical trees (Tian et al, 2003 ) and halophytes (Wang et al, 2007 , 2013 ; Yi et al, 2014 ) possibly by preventing the oxidization of polyphenol to polyquinones and the activity of many oxidizing enzymes under highly reduced conditions of the extraction buffer. Historically, BPP based protein extraction methods has been demonstrated as effective for the subcellular fractionation of laticifer latex in Hevea brasiliensis (Wang et al, 2010 ) and in the starch rich tuberous roots of Casava ( Manihot esculenta ; Wang D. et al, 2016 ) producing protein extracts compatible with 2-DE and MS.…”