Looking deep inside: detection of low-abundance proteins in leaf extracts of Arabidopsis and phloem exudates of pumpkin

Abstract

The field of proteomics suffers from the immense complexity of even small proteomes and the enormous dynamic range of protein concentrations within a given sample. Most protein samples contain a few major proteins, which hamper in-depth proteomic analysis. In the human field, combinatorial hexapeptide ligand libraries (CPLL; such as ProteoMiner) have been used for reduction of the dynamic range of protein concentrations; however, this technique is not established in plant research. In this work, we present the application of CPLL to Arabidopsis (Arabidopsis thaliana) leaf proteins. One- and two-dimensional gel electrophoresis showed a decrease in high-abundance proteins and an enrichment of less abundant proteins in CPLL-treated samples. After optimization of the CPLL protocol, mass spectrometric analyses of leaf extracts led to the identification of 1,192 proteins in control samples and an additional 512 proteins after the application of CPLL. Upon leaf infection with virulent Pseudomonas syringae DC3000, CPLL beads were also used for investigating the bacterial infectome. In total, 312 bacterial proteins could be identified in infected Arabidopsis leaves. Furthermore, phloem exudates of pumpkin (Cucurbita maxima) were analyzed. CPLL prefractionation caused depletion of the major phloem proteins 1 and 2 and improved phloem proteomics, because 67 of 320 identified proteins were detectable only after CPLL treatment. In sum, our results demonstrate that CPLL beads are a time- and cost-effective tool for reducing major proteins, which often interfere with downstream analyses. The concomitant enrichment of less abundant proteins may facilitate a deeper insight into the plant proteome.

Figure 1.

1D-SDS-PAGE of CPLL fractionation of Arabidopsis leaf proteins. Ten milligrams of leaf proteins was incubated with CPLL according to the manufacturer’s instructions. After four washing steps, proteins were eluted from the CPLL beads. Aliquots of the different fractions were separated by SDS-PAGE, and proteins were stained with Sypro Ruby. The relative masses of protein standards are indicated on the left. C, Crude leaf extract; CPLL, eluate; FT, flow through; W1 to W4, wash fractions. The position of the large subunit of Rubisco is marked with an arrow.

Figure 2.

2D-DIGE analysis of the effect of CPLL fractionation on the Arabidopsis leaf proteome. Each 50-µg protein of crude extract and CPLL eluate was stained with two different probes and separated on the same gel by 2D-DIGE, resulting in strikingly different spot patterns. The top shows spot patterns for both samples separately (DIGE modus black/white). Boxed areas depict selected proteins enriched upon CPLL treatment. The bottom shows a 2D-DIGE fluorescent overlay image of the above CPLL-treated (green) and crude extract (red) samples. A pH gradient of 4 to 7 was used for isoelectric focusing. Results are representative of four independent 2D-DIGE experiments.

Figure 3.

The effect of CPLL treatment on Arabidopsis leaf proteins. Leaf extracts were treated with CPLL according to the manufacturer’s protocol. The protein composition of the CPLL eluate was analyzed by LC-MS/MS and compared with the protein composition of the corresponding protein crude extract. A total of 1,284 unique proteins were identified in the control sample. Two hundred (15%) additional proteins could be identified by using CPLL for extraction.

Figure 4.

Analysis of a pH-based CPLL fractionation of Arabidopsis leaf proteins by 1D-SDS-PAGE. Protein extracts were adjusted to pH 4, 7, and 9 before application of CPLL. After CPLL fractionation, 15 µg of each fraction was separated by 1D-SDS-PAGE. Proteins were stained with Sypro Ruby. The relative masses of protein standards are indicated on the left. E4, E7, and E9, Protein extract at pH 4, 7, and 9; L4, L7, and L9, corresponding CPLL-treated proteins. The position of Rubisco is marked with an arrow.

Figure 5.

Identification of Arabidopsis leaf proteins by LC-MS/MS after the application of CPLL beads at three different pH values, and elution from the beads using hot SDS/DTT. The protein composition of CPLL eluates captured at pH 4, 7, and 9 was compared with the corresponding pH controls. In total, 1,704 proteins were detected. A total of 1,192 proteins were identified in control samples, whereas 512 (43%) additional proteins were found exclusively after treatment with CPLL at three different pH values.

Figure 6.

Analysis of CPLL fractionation of pumpkin phloem proteins. Phloem crude extracts and CPLL fractions (pH 5.2 and 7.8) were separated by 1D-SDS-PAGE. Loaded CPLL eluates were equivalent to 30 µL of phloem exudate. The two major phloem proteins PP1 (96 kD) and PP2 (24 kD) are marked with stars.

Figure 7.

Comparison of the number of identified proteins in crude phloem exudates and CPLL-treated exudates of pumpkin. LC-MS/MS analyses led to the identification of 320 phloem proteins, of which 253 proteins were found in untreated control samples. However, 67 additional proteins could be exclusively detected by using CPLL beads.