**1. Introduction**

Potato storage proteins provide necessary nutrients for the development of tuber, mature-tosprouting tuber transition and successful plant growth [1–4]. The patatin is the major VSP of

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*S. tuberosum*, accounting for up to 45% of the total soluble protein [1, 4–7]. However, the molecular mechanism that triggers the cleavage of the patatin along the tuber life cycle has not to date been identified. The regulatory mechanisms involved in the synthesis and degradation of storage proteins are better known in dry seeds [8–13]. Phosphorylation has proven to be a key regulator mechanism in the maturation, dormancy and germination of seed storage proteins (SSPs). Thus, reverse phosphorylation of the phytohormone abscisic acid (ABA) seems to play a crucial regulatory role in the synthesis of SSPs at the transcriptional level [11, 12]. More specifically, phosphoproteome studies in rapeseed and rice reported that cruciferins and cupins achieve higher levels of phosphorylation at the late maturation stage [9, 13]. In addition, mobilization of the major SSP in the common bean, phaseolin, was found to occur in germinating seeds through degradation of highly phosphorylated isoforms [10]. It suggests that degradation of SSPs in dry-to-germinating seed transition occurs through a phosphorylation-dependent regulatory mechanism.

The past few years have witnessed a steady discovery of phosphorylated SSPs such as cruciferins, napins, cupins, legumins and vicilins [8–10, 14, 15], but no evidence of phosphorylated isoforms has been reported to date in patatin or other VSPs such as sporamins and ocatins. Therefore, elucidating the question of whether patatin can be phosphorylated is a mandatory initial step in follow-up research concerning the molecular processes underlying its mobilization. First of all, the term patatin applies to a group of glycoproteins encoded by a gene family constituted by ~10–18 genes per haploid genome, most of them organized as a single gene cluster at the end of the long arm of chromosome 8 [16–18]. Patatin gene family members exhibit a very high degree of nucleotide sequence identity [19, 20]. In addition, patatin is a family of immunologically indistinguishable isoforms with similar structural properties and thermal conformational stability [21, 22]. Overall, extensive heterogeneity in molecular mass (40–45 kDa) and isoelectric point (4.5–5.2) seems to be the most salient differential molecular features among isoforms [21–25].

The 2-DE has provided the most complete information about the heterogeneity in molecular mass (*M*<sup>r</sup> ) and isoelectric point (p*I*) of the patatin [23–25]. Specifically, a total of 17–23 spots with variations in *M*<sup>r</sup> and/or p*I* were detected in 2-DE patatin profiles obtained from different potato cultivars [25]. Variations in *M*<sup>r</sup> seem to be mainly due to differential N-glycosylation at three specific asparagine residues of the amino acid sequence by *N*-linked oligosaccharide side chains or glycans [21, 22, 25]. Charge differences among isoforms could be explained by variations in positively and negatively charged amino acids [22]. However, variable phosphorylation has potential to change the p*I* of proteins by substituting hydroxyl groups on amino acid residues with negatively charged phosphate groups [26]. Therefore, phosphorylation could be a plausible but unexplored factor contributes to explain charge heterogeneity among patatin isoforms on 2-DE gels.

In this study, we undertook a proteomic approach addressed to the identification and mapping of phosphorylated isoforms of the patatin multigene family based on highresolution 2-DE. First, relatively abundant tuber proteins were successfully separated from low-abundance proteins by loading low amounts of total protein sample into 2-DE gels. Subsequently, high-abundance patatin proteins were identified and distinguished from other tuber abundant proteins on 2-DE gels using mass spectrometry (MS) techniques. Second, direct and rapid in-gel multiplex identification and mapping of phosphorylated isoforms of the patatin were achieved using the Pro-Q Diamond phosphoprotein stain (Pro-Q DPS), which specifically binds to the phosphate moieties of phosphoproteins [27]. Third, quantitative profiling of phosphorylated patatin isoforms was assessed by chemical dephosphorylation of phosphoproteins with hydrogen fluoride-pyridine (HF-P) [28–30]. For this purpose, the volume difference between phosphorylated and dephosphorylated 2-DE patatin spots was used to quantify protein phosphorylation levels. This experimental pipeline is a highly valuable top-down proteomic approach for the identification and mapping of phosphorylated isoforms of high-abundance storage proteins. It has been instrumental in unravelling the quantitative profiling of phosphorylated phaseolin isoforms in common bean seeds as well as their dynamic changes in dry-to-germinating seed transition [10]. Proteomic analyses were performed from total protein extracts of mature tubers of cultivar Kennebec. The obtained results will facilitate follow-up studies for better understanding of the regulatory mechanisms underlying patatin degradation and its biochemical status along the tuber life cycle.
