Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two-dimensional gel electrophoresis.
BACKGROUND
A variety of sample preparation protocols for plant proteomic analysis using two-dimensional gel electrophoresis (2-DE) have been reported. However, they usually have to be adapted and further optimised for the analysis of plant species not previously studied.


OBJECTIVE
This work aimed to evaluate different sample preparation protocols for analysing Carica papaya L. leaf proteins through 2-DE.


METHODS
Four sample preparation methods were tested: (1) phenol extraction and methanol-ammonium acetate precipitation; (2) no precipitation fractionation; and the traditional trichloroacetic acid-acetone precipitation either (3) with or (4) without protein fractionation. The samples were analysed for their compatibility with SDS-PAGE (1-DE) and 2-DE. Fifteen selected protein spots were trypsinised and analysed by matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS), followed by a protein search using the NCBInr database to accurately identify all proteins.


RESULTS
Methods number 3 and 4 resulted in large quantities of protein with good 1-DE separation and were chosen for 2-DE analysis. However, only the TCA method without fractionation (no. 4) proved to be useful. Spot number and resolution advances were achieved, which included having an additional solubilisation step in the conventional TCA method. Moreover, most of the theoretical and experimental protein molecular weight and pI data had similar values, suggesting good focusing and, most importantly, limited protein degradation.


CONCLUSIONS
The described sample preparation method allows the proteomic analysis of papaya leaves by 2-DE and mass spectrometry (MALDI-TOF-MS/MS). The methods presented can be a starting point for the optimisation of sample preparation protocols for other plant species.
Rodrigues
Silas Pessini
SP
Instituto de Bioquímica Médica, Unidade de Espectrometria de Massas e Proteômica e Rede Proteômica do Rio de Janeiro, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Ventura
José Aires
JA
Zingali
R B
RB
Fernandes
P M B
PM
D029441:Carica / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D018515:Plant Leaves / Q000737:chemistry*; D010940:Plant Proteins / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010940:Plant Proteins