Initially, an isopropanol method was developed to deplete glycinin and β-conglycinin subunits from total soybean seed proteins. Authors used 10–80% isopropanol in water to preferentially deplete the seed SPs (Natarajan et al., 2009). Of these concentrations tested, 30–60% isopropanol showed sufficient depletion of both glycinin and β-conglycinin subunits. Further resolution of isopropanol extracted proteins showed that 40% isopropanol had the maximum number of LAP spots as compared to other concentrations (30, 50, and 60%) (Natarajan et al., 2009). Recently, it was shown that ultrasonic treatment during isopropanol extraction increases the amount of LAPs. A 26.42% increase in the LAPs was observed when soybean seed proteins were isolated with 50% isopropanol along with the ultrasonic (400 W) treatment for 10 min (Liu et al., 2016).

The acid-SDS based extraction method was developed specifically to remove SPs from Pinellia ternata tubers, which are widely used in Chinese traditional medicines (Wu et al., 2012). Here, the property of differential solubility of SPs in acids was exploited to remove the most abundant tuber proteins. This method involves extraction of SPs in 10% acetic acid followed by extraction of soluble proteins in SDS-based buffer (0.5 M Tris-HCl, pH 8.8, 2% SDS, and 20 mM DTT). Initially, different concentrations of acetic acid (1, 5, 10, 30, and 60%) were tested of which 10% was found as the optimal concentration for the depletion of major SP of 25 kDa. After acetic acid extraction/precipitation, the pellet thus obtained, was washed twice with chilled acetone to remove the residual acetic acid and then solubilized in a SDS-based buffer. Proteins from the SDS buffer were recovered using phenol-methanolic ammonium acetate precipitation where samples were mixed with an equal volume of phenol. After centrifugation, the lower phenol phase was mixed with five volumes of 0.1 M ammonium acetate in methanol and incubated at -20°C for 2 h to precipitate the proteins. The pellet thus obtained was dissolved in either SDS-PAGE loading buffer or 2D rehydration buffer and directly loaded onto the gels. The 1D and 2D gel profiles showed that 25 kDa SP was almost removed while the 11 kDa SP was enriched along with the LAPs in the pellet-fraction proteins, probably because of its lesser solubility in the acids. This method is unable to remove the acid insoluble SPs from the LAPs, which is one of the drawbacks of this protocol (Wu et al., 2012).

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. Post-CAPE, the 2D gels clearly showed the removal of vicilins from the total maize embryo proteins. 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).

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 MgCl₂ and 2% v/v β-mercaptoethanol] followed by incubation of total protein extract with 50% ethanol for 1 h at -20°C. Proteins from ethanol-pellet (EP) and -supernatant (ES) fractions, obtained after centrifugation, were isolated using the phenol precipitation method. The 1D and 2D gel profiles clearly showed a dose-dependent fractionation of SPs in the ES fraction and concurrently enrichment of LAPs in the EP fractions. Out of the different concentrations of ethanol tested (20–80%), 50% showed best results in terms of enrichment of LAPs in the EP fraction. A recent study used EPM to compare the anthocyanin biosynthesis in the tuberous roots of yellow and purple sweet potato cultivars (Wang et al., 2016). Increased abundance of starch phosphorylase and phosphoglucomutase was observed in purple cultivar, which indicated that starch degradation might provide higher substrates for anthocyanin biosynthesis in purple-colored as compared to the yellow-colored sweet potato cultivar (Wang et al., 2016). This study further supports the reproducibility and applicability of EPM for comparative proteome analysis.

The same research group that developed the isopropanol method, also developed a calcium-based method to deplete SPs of soybean seeds (Krishnan et al., 2009). However, the effect of this method on enrichment of LAPs was more pronounced than the previously reported isopropanol method and is applicable to the seeds of many other plants (Table 1). In this protocol, seed proteins were first isolated in an aqueous buffer (20 mM Tris-HCl, pH 6.8, containing protease inhibitor cocktail) and then incubated with 10 mM of CaCl₂ for 10 min with constant shaking followed by centrifugation. The 1D gel profile clearly showed the depletion of approximately 10 bands corresponding to the HAPs. Resolution of calcium-fractionated protein samples on 2-DE and difference in-gel electrophoresis (DIGE) further showed removal of 87 ± 4% of the HAPs, pronouncing the detection of 541 LAP spots. Phosphoprotein staining of the calcium-fractionated proteins on 2-D gels led to the detection of 63 new phosphorylated spots of which detection of 15 spots was enhanced in the calcium-fractionated samples, suggesting the depletion of SPs can be fruitful in the phosphoproteome analysis as well (Krishnan et al., 2009). Recently, the calcium precipitation method was utilized to investigate the Lys-N^𝜀-acetylome of developing soybean seeds, further confirming the applicability of this method for post-translational modification analysis (Smith-Hammond et al., 2014).

Protamine sulfate precipitation (PSP) method was first developed to precipitate the RuBisCO protein from the leaves of green plants where it was shown that addition of 0.1% protamine sulfate (PS) specifically depletes the RuBisCO in pellet fraction (Kim et al., 2013) (Gupta and Kim, 2015). The application of this method was extended to seed proteomics where it was shown that incubation of seed protein extract with 0.05% PS significantly depleted the SPs of soybean, broad bean, pea, and wild soybean in pellet fraction. In the case of peanut, 0.1% PS was required to differentially fractionate the SPs. Briefly, the proteins were first isolated in the Tris-Mg/NP-40 extraction buffer [0.5 M Tris-HCl (pH 8.3), 2% v/v NP-40, 20 mM MgCl₂] followed by incubation with required concentration of PS (0.05–0.1%) for 30 min on ice. Finally, supernatant was clarified by centrifugation and PS-supernatant was used as LAPs rich fraction as SPs depleted into the pellet fraction (Kim et al., 2015). Recently, the PSP method was used for the comparative analysis of soybean seeds differing in total protein and oil contents, highlighting the efficacy of PSP method in comparative proteome analysis (Min et al., 2015).

Similar to the PSP method, PEG method was initially developed to remove the RuBisCO protein from the rice leaves (Kim et al., 2001). Recently, Wang et al. (2015) found that sequential fractionation of lettuce seed proteins with increasing percentage of PEG showed significant depletion of SPs. Using three different concentrations (4, 8, and 12%), authors found 8% as an effective PEG concentration. Addition of 4% PEG led to the depletion of approximately 55% of total proteins, majority of which were identified as SPs. Moreover, addition of 8% PEG in the supernatant fraction further precipitated the SPs in the pellet fraction (Wang et al., 2015). A total of 133 more spots were visualized in the 2D gel of 8% PEG fractionated sample as compared to total seed sample, indicating the potency of PEG method in the enrichment of LAPs. Using the PEG fractionation method, authors showed the involvement of mevalonate pathway in the germination and thermo-inhibition of lettuce seeds.

Many of the SPs, including β-conglycinin of soybean seed and 25 kDa SP of P. ternata tubers, are N-glycosylated in nature. Therefore, an attempt was also made to use lectin affinity chromatography for the removal of β-conglycinin and other glycosylated proteins from the total soybean seed proteins (Miernyk and Johnston, 2006). Total seed proteins were isolated using the phenol-methanolic ammonium acetate precipitation method and then dissolved in 8 M urea, 10% glycerol, and 0.5% (v/v) Triton X-100. Isolated proteins were loaded onto a Con A-Sepharose column and after washing off the unbound proteins, glycosylated proteins were eluted using 400 mM α-methyl mannoside. The 1D and 2D gel profiles showed significant removal of β-conglycinin and a 26 kDa polypeptide from the unbound proteins. However, one of the major demerits of this protocol was that some of the low abundant glycosylated proteins can also be lost along with abundant glycosylated proteins while other non-glycosylated abundant proteins can be eluted out in flow-through fraction.

Combinatorial Peptide Ligand Library (CPLL) is one of the most promising technologies for the enrichment of the hidden proteome (Boschetti and Righetti, 2013) and (Righetti and Boschetti, 2016). CPLL has the potential to reduce the concentration of a wide range of HAPs and is compatible to any sample/tissue type. CPLL columns consist of several million hexapeptides that have been generated using 16 different amino acids. These hexapeptides are designed in such a way that these can recognize almost all the proteins present in a sample (Righetti and Boschetti, 2016). When the total protein extract is loaded onto a CPLL column, these proteins bind with their hexapeptide partner beads. HAPs saturate their partner beads first and thus their major fraction remain unbound which is finally washed out as the flow through. On the other hand, when additional protein extract is loaded onto a CPLL column, LAPs keep on binding with their partner beads and thus get enriched, which can then be analyzed by downstream gel-based or gel-free proteomic approaches (Righetti and Boschetti, 2016). Esteve and co-workers utilized the CPLL technology for analyzing the olive seed proteome (Esteve et al., 2012). Following CPLL, 31 more seed proteins including SPs, oleosins and histones, were identified, indicating the efficacy and applicability of CPLL technique in seed proteomics.