In the ever-evolving realm of agricultural research, the spotlight intensifies on the dynamic interplay between plants and their environment. Jerusalem artichoke (Helianthus tuberosus L.), an annual herbaceous plant renowned for its robustness in challenging conditions, emerges as a pivotal player in this intricate dance. Thriving across diverse soils and temperatures, from arid to saline-alkaline and cold to hot, this plant has become a global cultivation phenomenon.
Beyond its adaptability, Jerusalem artichoke holds significance in various industries, serving as a source of raw materials, functional foods, biomass, and contributing to biomedical and chemical endeavors. A dedicated research team delves into the cultivation and utilization of Jerusalem artichoke tubers, exploring their potential as a traditional or alternative medicine. The primary focus lies in understanding the protein composition of these tubers, with a particular emphasis on their healing properties related to diabetes.
Case 1 Label Free Quantitative Proteomics Reveals the Proteome of Kikuyu (Helianthus tuberosus L.) Tubers
Helianthus tuberosus L., commonly known as Jerusalem artichoke (JA), is an annual herbaceous plant in the Asteraceae family. Renowned for its resilience to environmental stress, this plant thrives in diverse soils (ranging from dry to saline-alkaline) and temperatures (from cold to hot), making it cultivated in various regions worldwide. Besides its adaptability, this plant is notable for its significance in raw materials, functional foods, biomass, biomedical, and chemical industries. The research team has been devoted to cultivating and utilizing Jerusalem artichoke tubers as a traditional/alternative medicine in daily life, particularly focusing on lowering blood glucose levels and HbA1c in human subjects. Understanding the protein composition of the tubers may unveil their healing properties, especially those related to diabetes. This study employed three commercially processed JA tuber products (powder and slices) for label-free quantitative proteomic analysis.
The label-free quantitative proteomic approach was utilized to analyze differential protein abundance among the three tuber samples. After removing contaminants and low-confidence proteins, a total of 2967 high-confidence protein groups were identified (Figure 1 A). Due to the use of three different tuber samples, multiple missing values were expected after MS runs; thus, two replicates with repeatable identified protein groups in two out of three replicates (2102 protein groups) were selected for further analysis (Figure 1 A).
A Venn diagram analysis revealed that 1702 protein groups were commonly identified in all three tuber samples, while 89, 36, and 54 protein groups were exclusively identified in tubers 1, 2, and 3, respectively. Additionally, tubers 1 and 2 shared 112 protein groups, indicating a closer correlation between their protein compositions compared to pairs (2,3) and (3,1) (Figure 1 B). To assess the reproducibility of label-free protein quantification in triplicate for the same sample, a multi-scatter plot was generated using Perseus software (Figure 1 C). Scatter plots for the same sample exhibited a typical non-uniform distribution, being broader at lower intensities and narrower at higher intensities. The Pearson correlation coefficients for different replicates of the same sample were greater than 0.944, indicating a high correlation among the three replicates of the same sample. Histograms illustrated protein counts before and after missing value imputation (Figure 1 D).
Figure 1. Overview of Proteomic Differences in Three Different Jerusalem Artichoke (JA) Tubers (1, 2, 3)
Hierarchical clustering analysis divided differentially expressed proteins into four clusters, each exhibiting distinct expression profiles (Figure 2 A). Cluster 1 comprised 61 proteins primarily downregulated in tuber 3, while the 891 proteins in Cluster 1 were predominantly upregulated in tuber 2. Cluster 3 displayed 379 proteins with increased abundance in tuber 3, and Cluster 4, with 771 proteins, showed the highest abundance in tuber 1. A heatmap illustrating the abundance patterns of the top 48 differentially regulated proteins is presented in Figure 2 B. Among these 48 DEs, various proteins with unique functions were identified. Nineteen, 24, and 34 DE proteins were highly regulated, with 27, 22, and 14 proteins decreased in tubers 1, 2, and 3, respectively. The results indicate significant differences in protein content between tubers 1 and 3, while tuber 2 results are more diverse. Furthermore, Partial Least Squares Discriminant Analysis (PLS-DA) separated tubers 1 and 3 in Component 1, accounting for 32.7% of the total variance, and tuber 2 from tubers 1 and 3 in Component 2, representing 25.5% of the total variance (Figure 2 C). Finally, the top 15 proteins contributing to the separation in the PLS-DA plot were identified (Figure 2 D).
Figure 2. Statistical Analysis of JA Tuber Proteome
Following statistical analysis, the authors conducted functional classification based on Gene Ontology (GO) for the 2102 proteins, and PANTHER 19.0 detected 2044 proteins in the Helianthus annuus database. Subsequently, PANTHER pathway analysis was performed to categorize proteins into pathways, revealing that the majority were attributed to the ubiquitin-proteasome system and purine biosynthesis, followed by the TCA cycle, glycolysis, ketone body metabolism, cell cycle, and pyrimidine ribonucleotide pathways.
The study also identified several proteins directly or indirectly involved in various disease-related mechanisms. Three proteins were implicated in Alzheimer's disease, and 11 proteins were found associated with Huntington's disease pathway. Additionally, the authors identified well-known proteins with therapeutic significance, utilized for treating chronic diseases such as diabetes and cancer when consumed as dietary protein sources or through oral supplements. Several antimicrobial proteins were also identified (Figure 3).
Figure 3. GO Pathway Analysis of Tubers Proteome Using PANTHER Tool
This study employed high-throughput untargeted LC-MS/MS proteomic techniques to investigate the tuber proteome derived from two different processing methods of Helianthus tuberosus, including dry powder (Tuber 1 and 2) and dry slice (Tuber 3). The identified proteins and their classifications corroborated with previously unpublished research data, suggesting functionalities such as the reduction of blood glucose levels, association with human glycated hemoglobin (Hb-A1c), prevention of dementia, and therapeutic effects on Huntington's disease.
Reference
- Bakku, Ranjith Kumar, et al. "Unravelling the Helianthus tuberosus L.(Jerusalem artichoke, Kiku-Imo) tuber proteome by label-free quantitative proteomics." Molecules 27.3 (2022): 1111.
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