Proteomics in globe artichoke: Protein extraction and sample complexity reduction by PEG fractionation

Abstract

Here, we report the first leaf proteome analysis for globe artichoke. Three protein extraction protocols were tested and a reproducible Mg/NP-40-based method was established. Ribulose-1,5-biphosphate carboxylase-oxygenase (RuBisCO) is a highly abundant leaf protein, and its presence masks co-localizing, less abundant proteins. To remove RuBisCO from the sample, and thereby improve spot resolution, a PEG fractionation approach was elaborated. 2-DE profiles of various PEG fractions showed that the fractionation procedure was successful in excluding most of the RuBisCO, allowing for the detection of many low-abundance proteins. Western blot analysis was able to confirm the reduction in RuBisCO content achieved by PEG fractionation. In all, 841 distinct protein spots were detected, and 40 of these, selected from the RuBisCO region of the 2-DE profile, were successfully identified by MS. A number of homologues of these proteins also co-localize with RuBisCO in Arabidopsis thaliana. Keywords IntroductionThe globe artichoke (Cynara cardunculus L. var. scolymus) is an important component of the Mediterranean rural economy, but its improvement through breeding has been rather limited and its genome organization remains largely unexplored. Globe artichoke is mainly used in human nutrition: its immature inflorescence (capitulum, or more commonly ''head'' or ''bud'') represents the edible part of the plant and is consumed fresh, canned or frozen. It is also a promising source of biopharmaceuticals [1], such as luteolin and mono-/ di-caffeoylquinic acids [2,3]. These compounds are responsible for its therapeutic effects, which include hepatoprotective and choleretic activity [4], protection against oxidative damage caused by free radicals [5], inhibition of cholesterol biosynthesis and prevention of arteriosclerosis and other vascular disorders [6,7], inhibition of HIV integrase [8,9] and antibacterial and antifungal activity [10]. Because of the current interest in nutraceuticals for human healthcare, there has been a growing focus on the identity of the genes and networks involved in secondary metabolism. Only a limited number of genes involved in phenylpropanoid and sesquiterpene lactone metabolism has been isolated so far [11,12]. Recently, a set of basic proteomic tools has been applied to purify a soluble peroxidase (artichoke leaves soluble peroxidase) from globe artichoke leaves [13], but its proteome as a whole has not been explored. Although molecular markers have been developed [14][15][16][17] and applied to produce a genetic map [18], the globe artichoke genome also remains poorly researched.The genome is fixed in time, but the proteome is very plastic, depending on tissue type, developmental stage and age, and is also strongly modulated by the environment. The differential response of related proteomes to the same set of biotic and abiotic factors allows the genetic mapping of expressed genes [19]. These comparisons can also identify ''candidate proteins'' involved in the determination of a particular phenotyp...

Proteomic Analysis of Lettuce Seed Germination and Thermoinhibition by Sampling of Individual Seeds at Germination and Removal of Storage Proteins by Polyethylene Glycol Fractionation

“…As shown in Figure 2, D and E, many proteins were masked in the region of the 2-D gel where the storage proteins dominated. This situation occurs also in other plant tissues, such as leaf, in which Rubisco is highly abundant Acquadro et al, 2009). PEG fractionation is recognized as a convenient and reproducible method for the removal of Rubisco and to increase the resolution of low-abundance proteins in leaf extracts of many species (Xi et al, 2006;Ahsan et al, 2007;Lee et al, 2007;Acquadro et al, 2009).…”

“…PEG fractionation was performed according to Acquadro et al (2009). A flow chart of the PEG fractionation procedure is shown in Figure 2A.…”

“…PEG is an excellent general reagent for differential precipitation of proteins, and PEG fractionation is simpler and more effective in removing high-abundance proteins (Juckes, 1971). This approach has been applied to effectively remove most of the Rubisco in globe artichoke (Cynara scolymus; Acquadro et al, 2009), tomato (Solanum lycopersicum; Ahsan et al, 2007), rice , and Arabidopsis (Xi et al, 2006) leaves. In addition, it increases the number of detectable protein spots and allows for the detection of many low-abundance proteins in these tissues.…”

“…about 50% of the leaf protein is Rubisco). To improve the detection of the lower abundance proteins in proteomic analyses, especially by two-dimensional gel electrophoresis (2-DE), approaches have been developed to deplete high-abundance proteins partly or completely by precipitation during sample preparation using chemical compounds such as CaCl 2 , Ca 2+ phytate , and polyethylene glycol (PEG; Kim et al, 2001;Xi et al, 2006;Acquadro et al, 2009). PEG is an excellent general reagent for differential precipitation of proteins, and PEG fractionation is simpler and more effective in removing high-abundance proteins (Juckes, 1971).…”

Estudo proteômico de variedades de milho (<i>Zea mays</i> L.) obtidas por melhoramento clássico e por recombinação genética

“…A partir dessas diferenças mínimas foi possível distinguir as amostras com qualidade proteica melhorada (QPM) da amostra convencional normal (CN). Estudos prévios também realizaram géis 1-DE para avaliar a extração e perfil proteico de suas amostras quanto ao PM (ACQUADRO et al, 2009;NOGUEIRA et al, 2012;WU et al, 2011). …”