Seed storage proteins play a fundamental role in plant reproduction and human nutrition. They accumulate during seed development as reserve material for germination and seedling growth and are a major source of dietary protein for human consumption. Storage proteins encompass multiple isoforms encoded by multi-gene families that undergo abundant glycosylations and phosphorylations. Two-dimensional electrophoresis (2-DE) is a proteomic tool especially suitable for the characterization of storage proteins because of their peculiar characteristics. In particular, storage proteins are soluble multimeric proteins highly represented in the seed proteome that contain polypeptides of molecular mass between 10 and 130 kDa. In addition, high-resolution profiles can be achieved by applying targeted 2-DE protocols. 2-DE coupled with mass spectrometry (MS) has traditionally been the methodology of choice in numerous studies on the biology of storage proteins in a wide diversity of plants. 2-DE-based reference maps have decisively contributed to the current state of our knowledge about storage proteins in multiple key aspects, including identification of isoforms and quantification of their relative abundance, identification of phosphorylated isoforms and assessment of their phosphorylation status, and dynamic changes of isoforms during seed development and germination both qualitatively and quantitatively. These advances have translated into relevant information about meaningful traits in seed breeding such as protein quality, longevity, gluten and allergen content, stress response and antifungal, antibacterial, and insect susceptibility. This review addresses progress on the biology of storage proteins and application areas in seed breeding using 2-DE-based maps.

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The role of the protein phosphorylation mechanism in the mobilization of vegetative storage proteins (VSPs) is totally unknown. Patatin is the major VSP of the potato (Solanum tuberosum L.) tuber that encompasses multiple differentially phosphorylated isoforms. In this study, temporal changes in the phosphorylation status of patatin isoforms and their involvement in patatin mobilization are investigated using phosphoproteomic methods based on targeted two-dimensional electrophoresis (2-DE). High-resolution 2-DE profiles of patatin isoforms were obtained in four sequential tuber life cycle stages of Kennebec cultivar: endodormancy, bud break, sprouting and plant growth. In-gel multiplex identification of phosphorylated isoforms with Pro-Q Diamond phosphoprotein-specific stain revealed an increase in the number of phosphorylated isoforms after the tuber endodormancy stage. In addition, we found that the phosphorylation status of patatin isoforms significantly changed throughout the tuber life cycle (P < 0.05) using the chemical method of protein dephosphorylation with hydrogen fluoride-pyridine (HF-P) coupled to 2-DE. More specifically, patatin phosphorylation increased by 32% from endodormancy to the tuber sprouting stage and subsequently decreased together with patatin degradation. Patatin isoforms were not randomly mobilized because highly phosphorylated Kuras-isoforms were preferably degraded in comparison to less phosphorylated non-Kuras isoforms. These results lead us to conclude that patatin is mobilized by a mechanism dependent on the phosphorylation status of specific isoforms.

Patatin is the major tuber storage protein constituted by multiple isoforms highly variable across potato (S. tuberosum) varieties. Here, we report a first association study of the variability of patatin isoforms between cultivars with their differences in tuber quality traits. Patatin-based proteomic distances were assessed between 15 table and/or processing potato cultivars from profiles of patatin obtained by two-dimensional electrophoresis. The content of ash, dry matter, reducing sugars, starch, total protein, and amino acid composition was also evaluated in tubers of each cultivar. Results showed that proteomic distances were significantly (P < 0.05) associated with differences in the content of ash, dry matter, and essential amino acids. Proteomic distances were also able to identify outlier cultivars regarding the content of dry matter, content of protein, and protein quality. In conclusion, patatin-based proteomic distances can shorten the screening and selection processes of potato cultivars with advantageous characteristics in molecular breeding.

Protein phosphorylation is a reversible post-translational modification (PTM) with major regulatory roles in many cellular processes. However, the analysis of phosphoproteins remains the most challenging barrier in the prevailing proteome research. Recent technological advances in two-dimensional electrophoresis (2-DE) coupled to mass spectrometry (MS) have enabled the identification, characterization, and quantification of protein phosphorylation on a global scale. Most research on phosphoproteins with 2-DE has been conducted using phosphostains. Nevertheless, low-abundant and low-phosphorylated phosphoproteins are not necessarily detected using phosphostains and/or MS. In this study, we report a comparative analysis of 2-DE phosphoproteome profiles using Pro-Q Diamond phosphoprotein stain (Pro-Q DPS) and chemical dephosphorylation of proteins with HF-P from longissimus thoracis (LT) muscle samples of the Rubia Gallega cattle breed. We found statistically significant differences in the number of identified phosphoproteins between methods. More specifically, we found a three-fold increase in phosphoprotein detection with the HF-P method. Unlike Pro-Q DPS, phosphoprotein spots with low volume and phosphorylation rate were identified by HF-P technique. This is the first approach to assess meat phosphoproteome maps using HF-P at a global scale. The results open a new window for 2-DE gel-based phosphoproteome analysis.