Application of an improved proteomics method for abundant protein cleanup: molecular and genomic mechanisms study in plant defense

Abstract

High abundance proteins like ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) impose a consistent challenge for the whole proteome characterization using shot-gun proteomics. To address this challenge, we developed and evaluated Polyethyleneimine Assisted Rubisco Cleanup (PARC) as a new method by combining both abundant protein removal and fractionation. The new approach was applied to a plant insect interaction study to validate the platform and investigate mechanisms for plant defense against herbivorous insects. Our results indicated that PARC can effectively remove Rubisco, improve the protein identification, and discover almost three times more differentially regulated proteins. The significantly enhanced shot-gun proteomics performance was translated into in-depth proteomic and molecular mechanisms for plant insect interaction, where carbon re-distribution was used to play an essential role. Moreover, the transcriptomic validation also confirmed the reliability of PARC analysis. Finally, functional studies were carried out for two differentially regulated genes as revealed by PARC analysis. Insect resistance was induced by over-expressing either jacalin-like or cupin-like genes in rice. The results further highlighted that PARC can serve as an effective strategy for proteomics analysis and gene discovery.

Fig. 1.

Efficiency of Rubisco removal by PEI (Polyethyleneimine) precipitation. A, PEI precipitation of total soluble protein isolated by soluble protein extraction buffer. From the left to the right, lane 1 shows the total soluble protein extracted by soluble protein extraction buffer. Lane 2 to 5 shows the supernatant of soluble protein after precipitation with 10, 25, 50, and 100 mg/g PEI (weight/total protein), respectively. Lane 6 and 7 are dissolved protein pellets after PEI precipitation. B, Incompatibility between PEI precipitation and total protein extracted by Sigma Aldrich Plant Total Protein Extraction kit. From the left to the right, lane 1 shows the total protein extracted by the kit. Lane 2 and 3 shows total protein supernatant after PEI precipitation at 25 and 50 mg/g, respectively. PEI concentration (mg/g) is as indicated at the top. Molecular weight markers in kDa are shown on the left.

Fig. 2.

Flowchart of protein extraction, Rubisco removal and fractionation method. Rice protein samples were extracted and fractionized into five fractions. Aerial part of rice seedlings were ground into fine powder in liquid nitrogen. Total protein was extracted by Sigma Plant Total Protein Extraction Kit (TP). Total soluble protein TS and Insoluble protein (IS) were obtained by centrifugation after the powder was homogenized in extraction buffer. PEI precipitation was used to fractionize the TS fraction into Soluble Supernatant protein (SS) and PEI Precipitated Soluble protein (PS).

Fig. 3.

Rubisco removal efficiency evaluated by proteomics. The pie charts showed the percentage of spectral counts for Rubisco in total spectral counts identified in each fraction. A, TP. B, TS. C, IS. D, SS. E, PS.

Fig. 4.

Improvement of protein identification and differential protein expression analysis. A, Comparison of the number of protein identified by TS and the combination of SS and PS. B, Comparison of the number of protein identification for TP and the combination of homogenizations, SS, PS, and IS. C, Comparison of total number of combined proteins between 3 independent TP biological samples and 1 biological samples of SS, PS, and IS. D, Comparison of the number of differentially expressed proteins identified in FAW larvae treated and untreated rice samples with the same fractionations of B. The standard errors were calculated based on duplicates of the biological samples from different factions.

Fig. 5.

Gene Ontology analysis of differentially expressed protein. Pie charts shows GO distribution of (A) combination of SS, PS, and IS as well as (B) TP according to their biological processes.

Fig. 6.

Pathway analysis of up-regulated proteins by FAW herbivory as revealed by PARC. The figure focused on the carbohydrate related metabolisms.

Fig. 7.

qRT-PCR validation of mRNA expression for selected differentially expressed proteins. qRT-PCR validation of (A) Os12g14440 and (B) Os03g48770 gene expression after 0 h, 12 h, 24 h, and 48 h treatments. The relative expression levels as compared with the lowest expressing sample in the group were derived from modified ΔΔCT method. The average ratio and 95% confidence interval were derived from triplicate assays.

Fig. 8.

Comparison of leaf area damage caused by FAW larvae feeding on wild type and transgenic rice. The figure shows ANOVA analysis of three transgenic lines for both Os03g48770 (Cupin) and Os12g14440 (Jacalin) after FAW consumption. Wild type rice (group a) showed significant difference with transgenic lines (group b) (F_6,35 = 13.98; p < 0.01). The measurement is based on bioassay of 6 leave segments of each independent line. The average percentage of leaf area damage and 95% confidence intervals were presented.