Protein, phosphopeptides and phosphosites along 2-DE patatin spots of cv. Kennebec, identified from MALDI-TOF and MALDI-TOF/TOF MS data.
Protein phosphorylation plays a key role in the synthesis and degradation of dry seed storage proteins. In contrast, no evidence for phosphorylation has been reported to date in vegetative storage proteins (VSPs). The patatin multigene family encodes the major VSP of the potato, Solanum tuberosum L. This study addresses for the first time the identification and mapping of phosphorylated patatin forms based on high-resolution two-dimensional electrophoresis (2-DE) profiles. Patatin isoforms from mature tubers of cultivar Kennebec were separated by 2-DE and subsequently identified by tandem mass spectrometry. In-gel identification and mapping of phosphorylated isoforms were performed using the multiplex phosphoprotein-specific staining Pro-Q DPS. We found that phosphorylation is a ubiquitous post-translational protein modification associated with isoforms of patatin. In addition, protein dephosphorylation with hydrogen fluoride-pyridine coupled to 2-DE was used for quantitative profiling of phosphorylated patatin. This experimental approach showed that patatin comprises multiple isoforms with very different phosphorylation level. Thus, phosphorylation rates over isoforms ranged from 4.6 to 52.3%. Overall, the identification and mapping of differentially phosphorylated patatin opens up new exploratory ways to unravel the molecular mechanisms underlying its mobilization along the tuber life cycle.
- Solanum tuberosum
- seed storage proteins
- storage protein mobilization
- tuber phosphoproteome
- vegetative storage proteins
Potato storage proteins provide necessary nutrients for the development of tuber, mature-to-sprouting tuber transition and successful plant growth [1–4]. The patatin is the major VSP of
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 (
In this study, we undertook a proteomic approach addressed to the identification and mapping of phosphorylated isoforms of the patatin multigene family based on high-resolution 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 . 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 . 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.
2. Materials and methods
2.1. Plant material
Proteomic analyses were performed from mature potato tubers of cv. Kennebec (2n = 4x = 48). Larger pieces of lyophilized tuber were homogenized with a pre-cooled mortar and pestle. The samples were stored at −80°C until protein extraction. Four biological replicates were used for experiments.
2.2. Protein extraction and quantification
Total tuber proteins were extracted using the phenol extraction method. A 200 mg sample of lyophilized tuber was transferred to an extraction buffer (500 mM Tris-HCl, 500 mM EDTA, 700 mM sucrose, 100 mM KCl pH 8.0, 2% DTT and 1 mM PMSF). Tris-HCl (pH 6.6–7.9) saturated phenol was added and the phenol phase was collected using centrifuging (4500 rpm at 4°C). Protein precipitation solution of 0.1 M ammonium acetate in cold methanol was added. Protein pellet was washed with 0.1 M ammonium acetate and 10 mM DTT, and with 80% acetone and 10 mM DTT. The resuspended protein pellet was then diluted in lysis buffer (7 M urea; 2 M thiourea; 4% CHAPS; 10 mM DTT, and 2% PharmalyteTM pH 3–10, GE Healthcare, Uppsala, Sweden). Protein concentration was evaluated using the commercial CB-X protein assay kit (G-Biosciences, St. Louis, MO, USA) according to the instructions of the manufacturer for interfering agent removal and use with a microplate reader. The bovine serum albumin (BSA) was used as standard protein to generate calibration curves.
2.3. Two-dimensional electrophoresis (2-DE)
High-resolution 2-DE profiles of patatin isoforms were obtained following the procedure described in López Pedrouso et al. . Briefly, total protein samples (75 μg of protein) of each biological replicate were loaded into immobilized pH gradient (IPG) strips of 24-cm long and 4–7 pH linear gradient (Bio-Rad Laboratories, Hercules, CA, USA). First dimensional isoelectric focusing (IEF) was performed in a PROTEAN IEF Cell System (Bio-Rad Laboratories) after IPG strip rehydration for 12 h at 50 V. Rapid voltage ramping was subsequently applied to reach a total of 70 kVh. Equilibration of IEF strips was performed before running second dimension using equilibration buffers. The second dimension (SDS-PAGE) was performed on 10% polyacrylamide gels using an Ettan DALTsix large vertical electrophoresis system (GE Healthcare). Second-dimension gels were run using a constant electric current of 16 mA per gel for 15 h at 25°C.
2.4. Enzymatic deglycosylation of patatin
Patatin deglycosylation was performed with the enzyme protein-N-glycosidase F (PNGase F, New England Biolabs, Ipswich, MA, USA) according to the manufacturer specifications. A 75 μg sample of total protein extract from mature tuber was incubated with PNGase F (25 U/mL) and diluted in reaction buffer (New England Biolabs) until a final volume of 20 μL. The mixture was incubated for 12 h at 37°C. Patterns of deglycosylated patatin isoforms on 2-DE gels were obtained as described earlier.
2.5. Pro-Q staining for phosphoproteins
Pro-Q Diamond phosphoprotein stain (Pro-Q DPS, Molecular Probes, Leiden, The Netherlands) was used for the in-gel detection of phosphorylated patatin polypeptides, as described previously . Briefly, gels were fixed with 50% methanol and 10% acetic acid for 60 min and washed twice for 15 min each with distilled water. The gels were subsequently incubated for 120 min with two-fold water-diluted Pro-Q DPS, destained four times (30 min per wash) with 50 mM sodium acetate and 20% ACN pH 4.0, and washed again twice with distilled water (5 min per wash). The PeppermintStick™ (Molecular Probes) phosphoprotein marker was added to tuber protein extracts before 2-DE. Phosphorylated (ovalbumin, 45.0 kDa; and β-casein, 23.6 kDa) and unphosphorylated (β-galactosidase, 116.25 kDa; bovine serum albumin, 66.2 kDa; avidin, 18.0 kDa; and lysozyme, 14.4) PeppermintStick proteins were used as positive and negative controls of phosphorylation, respectively.
2.6. Chemical dephosphorylation of patatin
The chemical dephosphorylation of patatin was performed with hydrogen fluoride-pyridine (HF-P) as previously described [28, 29], with some modification . An amount of 1 mg of total protein extract from tuber of cv. Kennebec was dissolved in 250 μL of 70% HF-P and incubated on ice for 2 h. The mixture was neutralized by addition of 10 M sodium hydroxide solution. Proteins were desalinated using Amicon Ultra-4 centrifugal filter devices (Millipore, Billerica, MA, USA) and then eluted in 300 μL of lysis buffer. Prior to 2-DE, protein purification was performed using the Clean-up kit (GE Healthcare).
2.7. SYPRO Ruby staining for total protein
2-DE gels were stained with SYPRO Ruby fluorescent stain (Lonza, Rockland, ME, USA), for total protein density following the manufacturer’s indications. Pro-Q DPS-stained gels were also post-stained with SYPRO Ruby to detect total protein.
2.8. Image analysis
The 2-DE images from gels stained with Pro-Q DPS or SYPRO Ruby fluorescent dyes were acquired using a Gel Doc XR+ system (Bio-Rad Laboratories). Digitalized gels were analyzed with PDQuest Advanced software v. 8.0.1 (Bio-Rad Laboratories). Gel matching, spot identification and quantification of spot volumes were performed after background subtraction and normalization based on total density in valid spots. Automatic matches were manually checked. Only the reproducibly detected patatin spots across replicates were selected for quantitative analyses. Experimental p
2.9. In-gel digests
Protein spots of interest were excised from polyacrylamide gels and subjected to in-gel digestion with trypsin as described previously . Briefly, disulfide reduction and alkylation of the excised protein spots were performed with 10 mM DTT (Sigma-Aldrich, St. Louis, MO, USA) in 50 mM ammonium bicarbonate (Sigma-Aldrich) and 55 mM iodoacetamide (Sigma-Aldrich) in 50 mM ammonium bicarbonate, respectively. The gel pieces were washed with 50 mM ammonium bicarbonate in 50% methanol (HPLC grade, Scharlau, Barcelona, Spain), dehydrated with acetonitrile (ACN, HPLC grade) and subsequently dried in a SpeedVac (Thermo Fisher Scientific, Waltham, MA, USA). Dry gel pieces were incubated with modified porcine trypsin (Promega, Madison, WI, USA) at a concentration of 20 ng/μL in 20 mM ammonium bicarbonate, at 37°C for 16 h. After digestion, peptides were recovered by incubation (three times/20 min) in 40 μL of 60% ACN in 0.5% formic acid. The resulting tryptic peptides were concentrated in a SpeedVac and stored at −20°C.
2.10. Mass spectrometry (MS)
Protein identification was performed by MALDI-TOF and MALDI-TOF/TOF MS as reported by López-Pedrouso et al. . Peptides were dissolved in 4 μL 0.5% formic acid and then were mixed with an equal volume (0.5 μL) of matrix solution, containing 3 mg of α-Cyano-4-hydroxycinnamic acid (CHCA) dissolved in 1 mL of 50% ACN in 0.1% trifluoroacetic acid (TFA). The mixture was deposited using the thin layer method, onto a 384 Opti-TOF MALDI plate (Applied Biosystems, Foster City, CA, USA). Peptide MS and MS/MS data were acquired with a 4800 MALDI-TOF/TOF mass spectrometer (Applied Biosystems). MS spectra were acquired in positive-ion reflector mode with an Nd:YAG laser (355 nm wavelength) and an average number of 1000 laser shots. Each spectrum was internally calibrated with at least three trypsin autolysis products. All MS/MS spectra were performed by selecting precursors ions with a relative resolution of 300 full width at half maximum (FWHM) and metastable suppression. The 4000 Series Explorer Software v. 3.5 (Applied Biosystems) was used for mass data analysis. Combined peptide mass fingerprinting (PMF) and MS/MS fragment-ion spectra were interpreted with GPS Explorer Software v. 3.6 using Mascot software v. 2.1 (Matrix Science, Boston, MA, USA) to search against the
2.11. Data analysis
The phosphorylation rate at each spot was quantified using the measure
3. Results and discussion
3.1. Map of patatin isoforms based on 2-DE
Patatin isoforms in mature potato tuber of cv. Kennebec were first recognized on our 2-DE gels according to the previously reported studies on 2-DE patatin profiles [23–25]. We found that patatin profiles were constituted by a complex constellation of different spots showing large variations in
|Spot no.a||Exp. p||Match./cov. (%)c||Mascot score||Protein name (type)d||No. phosphopeptides/phosphosites|
The two-dimensional map of the patatin was implemented with the location of glycosylated isoforms using the enzyme PNGase F. It is an effective enzymatic method for removing almost all
3.2. In-gel identification of phosphorylated patatin isoforms
Pro-Q DPS was used for in-gel multiplex identification of phosphorylated patatin isoforms. Representative 2-DE images of patatin in mature tuber of cv. Kennebec on the same gel stained with Pro-Q DPS and post-stained with SYPRO Ruby are shown in Figure 2. The PeppermintStick markers used as positive and negative controls of protein phosphorylation validated the specificity of the recognition of phosphoproteins by Pro-Q DPS under our experimental conditions. It was found that all 20 patatin spots of the reference pattern exhibited Pro-Q DPS fluorescent signal. Similar result was obtained for patatins from mature tubers of cvs. Agria, Amanda and Ivory Russet (not shown). We can conclude, therefore, that phosphorylation is a ubiquitous PTM associated with isoforms of the patatin.
A prospective identification of phosphopeptides and phosphosites by MASCOT search using spectra data from MALDI-TOF and MALDI-TOF/TOF MS revealed 22 non-redundant patatin phosphopeptides containing 49 non-redundant phosphorylation sites (Table 1). Comparison with large-scale phosphoproteomic screens in other species using the Plant Protein Phosphorylation DataBase (P3DB)  suggests that most phosphorylation sites identified are novel to this study. Thus, no phosphorylated ortholog sites were identified in other plant phosphoproteomics data for
3.3. Quantitative profiling of phosphorylated patatin isoforms
Changes in the phosphorylation level across patatin spots of cv. Kennebec were assessed by chemical dephosphorylation of total tuber protein extracts with HF-P coupled to 2-DE. This experimental approach provides more efficient information than Pro-Q DPS to the identification and quantification of phosphorylated proteins on 2-DE gels . The reason is that the Pro-Q DPS fluorescent signal of spots containing low-abundance phosphopeptides is seriously suppressed by abundant non-phosphorylated phosphopeptides. The chemical dephosphorylation method has the advantage of using SYPRO Ruby stain, which combines good sensitivity with excellent linearity .
Representative 2-DE gel images of the patatin pattern before and after HP-F treatment are shown in Figure 3. First of all, note that spots of the protein phosphorylation marker, ovalbumin, underwent a basic shift on p
The phosphorylation level of each spot was evaluated with the measure
|Spot no.a||p||Mean (± SE) ||P ()b||95% bootstrap CI (CL, CU)c||99% bootstrap CI (CL, CU)c|
|1||4.84||39.75 ± 2.53||0.53||35.6, 44.1||34.6, 44.3|
|2||4.88||41.21 ± 6.07||0.57||31.9, 52.9||30.1, 54.5|
|3||4.90||43.05 ± 2.88||0.55||37.5, 47.9||37.4, 49.3|
|4||4.93||28.11 ± 4.35||0.76||23.8, 32.5||23.8, 32.5|
|5||4.96||42.83 ± 2.02||0.56||40.1, 46.9||39.4, 48.3|
|6||4.96||52.34 ± 4.10||0.57||46.4, 60.4||44.5, 61.8|
|7||5.02||39.01 ± 4.11||0.51||32.4, 45.6||30.4, 45.7|
|8||5.02||27.19 ± 5.61||0.52||16.9, 35.0||15.3, 35.0|
|9||5.05||32.48 ± 3.30||0.51||26.7, 37.3||22.9, 38.0|
|10||5.12||30.48 ± 4.72||0.57||23.3, 40.2||22.6, 42.4|
|11||5.12||25.13 ± 4.31||0.57||18.8, 33.1||16.6, 34.1|
|12||5.13||51.39 ± 5.52||0.55||41.2, 60.6||40.0, 62.1|
|13||5.14||4.60 ± 2.04||0.75||2.6, 6.6||2.6, 6.6|
|14||5.16||44.34 ± 9.37||0.52||25.7, 57.3||25.0, 59.0|
|15||5.20||41.87 ± 3.80||0.52||35.7, 49.3||34.7, 51.2|
|16||5.20||26.99 ± 8.37||0.58||18.1, 37.0||10.4, 37.0|
|17||5.23||35.96 ± 2.42||0.53||32.9, 40.9||32.2, 42.8|
|18||5.25||34.16 ± 4.09||0.53||26.2, 39.6||25.6, 40.0|
|20||5.27||11.95 ± 4.63||0.55||6.8, 20.9||5.6, 20.9|
Elucidating whether changes in abundance of protein phosphorylation reflect either changes in phosphorylation status or changes in the abundance of the protein itself is a major challenge in the interpretation of quantitative phosphoproteomics studies [39, 40]. Thus, phosphopeptide enrichment methods prior to high-resolution MS permit the identification of low-abundance phosphoproteins but prevent joint quantitation of phosphorylation status and abundance of proteins . However, our experimental approach allowed us to successfully tackle this problem. Thus, we have detected a statistically significant negative relationship between patatin spot volumes and their corresponding
The control of tuber sprouting is a major target in potato breeding because premature tuber sprouting during their lengthy storage leads to important quality and economic loss [41–43]. However, the molecular mechanisms controlling dormancy release and tuber sprouting are not yet sufficiently known [42–44]. The identification and mapping of phosphorylated isoforms of the patatin opens up new exploratory ways to unravel the molecular mechanisms underlying mobilization of VSPs. The finding of differentially phosphorylated isoforms is particularly relevant because increase (or decrease) in phosphorylation status without a parallel change in the amount of protein has been considered to be a useful indicator for a specific functional change [39, 40, 45]. In this regard, systematic follow-up studies on VSPs will be needed to assess whether their degradation takes place through a phosphorylation-dependent regulatory mechanism, as it occurs in common bean during dry-to-germinating seed transition . The establishment of a 2-DE-based reference map of patatin can be a very efficient tool to address this challenge in potato by monitoring changes in the phosphorylation status along the tuber life cycle.
This research was supported by funds from the Consellería do Medio Rural (Xunta de Galicia, Spain).