**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**

**2.3. Two-dimensional electrophoresis (2-DE)**

**2.4. Enzymatic deglycosylation of patatin**

were obtained as described earlier.

**2.5. Pro-Q staining for phosphoproteins**

**2.6. Chemical dephosphorylation of patatin**

gel for 15 h at 25°C.

68 Advances in Seed Biology

High-resolution 2-DE profiles of patatin isoforms were obtained following the procedure described in López Pedrouso et al. [10]. 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

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

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 [31]. 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

The chemical dephosphorylation of patatin was performed with hydrogen fluoride-pyridine (HF-P) as previously described [28, 29], with some modification [10]. 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

as positive and negative controls of phosphorylation, respectively.

purification was performed using the Clean-up kit (GE Healthcare).

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*I*-values over spots were determined using the linear scale of IEF-strips as reference, whereas *M*<sup>r</sup> -values were assessed using PeppermintStick (Molecular Probes) molecular weight markers and standard molecular mass markers ranging from 15 to 200 kDa (Fermentas, Ontario, Canada).

#### **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 [32]. 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. [10]. 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 *S. tuberosum* UniProtKB/ Swiss-Prot databases. Mascot database search parameters were: precursor mass tolerance of 50 ppm, MS/MS fragment tolerance of 0.6 Da, one missed cleavage allowed, carbamidomethyl cysteine (CAM) as fixed modification and oxidized methionine as variable modification. Identification of patatin phosphopeptides was also performed from spectrum data allowing phosphor-serine (PhosphoS), phosphor-tyrosine (PhosphoY) and phosphor-threonine (PhosphoT) residues as variable modification to search against the UniProtKB/Swiss-Prot databases. Analysis of phosphorylation sites was implemented using the Plant Protein Phosphorylation DataBase (P3 DB) [33]. All identifications and spectra were manually checked for validation. Proteins with at least two matched peptides and statistically significant (*p*-value < 0.05) MASCOT scores were selected as positively identified.

#### **2.11. Data analysis**

The phosphorylation rate at each spot was quantified using the measure *PR* [10]. It is defined as *PR* = [(*T* − *D*)/*T*] × 100, where *T* and *D* are the volumes of a given spot on 2-DE gels untreated (total protein volume) and treated (dephosphorylated protein volume) with HF-P, respectively. Non-parametric bootstrap confidence intervals (CIs) were obtained for mean values of *PR* across four biological replicates by the bias-corrected percentile method [34]. For each observed mean of *PR*, 2000 bootstrap samples of size N = 4 were drawn with replacement by applying a Monte Carlo algorithm. The 95 and 99% CIs for the observed mean of *PR* were constructed from distribution of 2000 bootstrap mean replications. The bootstrap estimate of bias was obtained from the proportion of bootstrap mean replications lower than the original estimate of the mean, and bias-corrected CIs were then calculated using the theoretical normal distribution as described by Efron [34]. *PR* data were clustered by using the unweighted pair-group method with arithmetic averaging (UPGMA). The UPGMA dendrogram derived from the matrix of pairwise *PR*-values was generated using NTSYSpc v. 2.1 software (Applied Biostatistics, Setauket, NY, USA). Descriptive statistics and Spearman's correlation test were calculated with the IBM SPSS Statistics 20 (SPSS, Chicago, IL, USA) statistical software package.
