Two-dimensional liquid chromatography technique coupled with mass spectrometry analysis to compare the proteomic response to cadmium stress in plants

Abstract

Plants are useful in studies of metal toxicity, because their physiological responses to different metals are correlated with the metal exposure dose and chemical state. Moreover a network of proteins and biochemical cascades that may lead to a controlled homeostasis of metals has been identified in many plant species. This paper focuses on the global protein variations that occur in a Populus nigra spp. clone (Poli) that has an exceptional tolerance to the presence of cadmium. Protein separation was based on a two-dimensional liquid chromatography technique. A subset of 20 out of 126 peaks were identified as being regulated differently under cadmium stress and were fingerprinted by MALDI-TOF. Proteins that were more abundant in the treated samples were located in the chloroplast and in the mitochondrion, suggesting the importance of these organelles in the response and adaptation to metal stress.

Figure 1

Virtual 2D maps created by the software “ProteoVue” for two independent untreated samples. Proteins were extracted as described in the Materials and Methods Section. Equal amounts of protein (1.3 mg) were loaded into the 1st dimension separation column (HPRP). This figure shows the isoelectric point (pI) fractions (from 4.22 to 5.9) for two independent biological replicates.

Figure 2

Magnified area of a virtual gel obtained after 2nd dimension separation (HPRC), for the 28th pH fraction, and reproduced by “DeltaVue” software. The protein pattern of untreated plants (0 μM CdSO₄) is shown in shades of blue on the left and the protein pattern of treated plants (50 μM CdSO₄) is shown in shades of yellow on the right. The center column demonstrates the differences in protein abundance between the control and the treated protein samples, as indicated by arrows at the peaks/ bands for proteins of different abundances.

Figure 3

A “ProteoVue” 2D map of a leaf protein extract from Populus nigra clone “Poli”, grown for 3 weeks in nutrient solution supplemented with 50 μM CdSO₄. The x-axis is in isoelectric point (pI) units from 4.0 to 8.0. The y-axis displays increasing hydrophobicity. The color scale of the bands represents the relative intensity of each band by UV detection at 214 nm. The proteins detected by MALDI/TOF analysis are numbered and highlighted with yellow arrows.