Branched Chain Amino Acids Analysis Service

What are Branched Chain Amino Acids?

Branched Chain Amino Acids (BCAAs) are essential amino acids that play critical roles in protein synthesis, muscle maintenance, and energy production within the human body. Comprising leucine, isoleucine, and valine, BCAAs are unique for their branched chemical structure, which distinguishes them from other amino acids.

Key Functions:

• Muscle Protein Synthesis: BCAAs are integral to the process of building and repairing muscle tissue, making them essential for athletes and individuals focused on physical fitness.
• Energy Production: During intense exercise or periods of fasting, BCAAs can be oxidized in muscle cells to provide energy, helping to sustain performance and support endurance.
• Regulation of Metabolism: Leucine, in particular, acts as a signaling molecule that stimulates muscle protein synthesis and regulates insulin sensitivity, contributing to overall metabolic health.

Research indicates that BCAA levels can influence various metabolic pathways and may impact conditions such as muscle wasting, metabolic disorders, and insulin resistance. Accurate measurement of BCAA levels is essential for understanding these metabolic processes and developing targeted nutritional strategies.

Branched Chain Amino Acids Analysis in Creative Proteomics

Quantification of BCAAs: Using High-Performance Liquid Chromatography (HPLC), we accurately quantify concentrations of leucine, isoleucine, and valine in various biological samples. This targeted metabolomics method ensures sensitivity and specificity, essential for studying metabolic pathways and assessing nutritional status.

Metabolic Pathway Analysis: Our services include detailed metabolic pathway analysis of BCAAs, elucidating their roles in energy metabolism, protein synthesis, and regulatory functions within the body. This analysis helps in understanding how BCAA levels influence overall metabolic health and athletic performance.

Customized Analytical Approaches: Creative Proteomics offers customized approaches tailored to specific research needs or clinical requirements. Whether investigating BCAA metabolism in disease states or optimizing nutritional interventions, our team provides tailored solutions to meet your objectives.

Comprehensive Reporting: We provide comprehensive technical reports detailing experimental procedures, analytical methodologies (including MS/MS instrument parameters), and precise quantification of BCAAs. Reports also include analytical metrics such as CVs (Coefficient of Variation), ensuring data reliability and reproducibility.

Expert Consultation: Our team of experienced scientists and bioanalytical experts offer consultation services to interpret results, discuss experimental design, and provide insights into BCAA analysis findings. This collaborative approach ensures that your research or clinical study achieves its objectives effectively.

Analytical Techniques for Branched Chain Amino Acids Analysis

High-Performance Liquid Chromatography (HPLC)

We employ state-of-the-art HPLC systems, such as the Agilent 1200 Series, for separating and quantifying BCAAs in biological samples. This system includes precise pumps for solvent delivery, an autosampler for automated sample handling, and a UV detector for sensitive detection.

Mass Spectrometry (MS/MS):

Our lab is equipped with Thermo Scientific TSQ Quantiva Triple Quadrupole Mass Spectrometers, which allow targeted analysis of BCAAs and their metabolites. This system features advanced ionization sources, triple quadrupole analyzers for precise ion selection and fragmentation, and sophisticated data analysis software for accurate quantification.

Supporting Equipment:

We utilize centrifuges for sample preparation, ultrafiltration units for sample cleanup, and calibration standards with isotopically labeled internal standards to ensure instrument accuracy and reliability.

List of Branched Chain Amino Acids Analysis

Essential Branched Chain Amino Acids (BCAAs)

Non-proteinogenic BCAAs

BCAA Metabolites

Sample Requirements for Branched Chain Amino Acids Analysis

Considerations:

Sample Collection: Ensure proper handling and collection techniques to avoid contamination and degradation.

Storage: Store samples at -80°C to maintain stability until analysis.

Labeling: Clearly label samples with unique identifiers and relevant information to ensure accurate analysis and data interpretation.

Special Requirements: Some samples may require special preparation steps (e.g., protein precipitation, filtration) before analysis.

Report

• A detailed technical report will be provided at the end of the whole project, including the experiment procedure, instrument parameters.
• Analytes are reported as uM or ug/mg (tissue), and CV's are generally<10%.
• The name of the analytes, abbreviation, formula, molecular weight and CAS# would also be included in the report.

Comparative metabolite profiling of salt sensitive Oryza sativa and the halophytic wild rice Oryza coarctata under salt stress

Journal: Plant‐Environment Interactions

Published: 2024

Background

The study aimed to investigate the metabolic responses of salt-sensitive Oryza sativa (BRRI dhan28) and salt-tolerant Oryza coarctata under salt stress conditions. Given the importance of understanding plant responses to environmental stressors like salinity, the research focused on comparing how these two species adapt at the metabolite level, potentially uncovering metabolic markers of salt tolerance.

Materials & Methods

1. Plants Growth Condition and Treatment

The experiment was conducted at the Plant Biotechnology Laboratory, University of Dhaka. O. sativa seeds were prepared by washing, soaking, and incubating before placement in trays with Yoshida's solution. O. coarctata, which propagates vegetatively, was treated similarly. Salt stress (increasing from 60 mM to 120 mM NaCl) was gradually applied to mimic natural conditions and prevent shock. Controls received no salt.

2. Metabolite Extraction

Root samples from both species and conditions were extracted using 80% methanol according to the protocol provided by Creative Proteomics Inc. Extracts were prepared in triplicate. The samples were sonicated, vortexed, and centrifuged, and the supernatant was collected and stored at -80°C before drying. Processed samples were shipped to Creative Proteomics, Inc for analysis of the total untargeted metabolites.

3. Targeted Metabolomic Analysis

For targeted metabolomic analysis, specific metabolites of interest, such as amino acids, organic acids, and lipids, were quantified using a targeted LC-MS/MS approach. Samples were prepared and analyzed in triplicate to ensure accuracy and reproducibility.

• LC-MS Analysis

The separation was performed by ACQUITY UPLC combined with Q Exactive MS and screened with ESI–MS. The LC system used a gradient elution with solvents A (0.05% formic acid water) and B (acetonitrile). The flow rate was 0.3 mL·min−1, with the column temperature at 40°C and the sample manager temperature at 4°C.

Mass spectrometry parameters:

• ESI+: Heater Temp 300°C; Sheath Gas Flow rate, 45 arb; Aux Gas Flow Rate, 15 arb; spray voltage, 3.0KV; Capillary Temp, 350°C; S-Lens RF Level, 30%.
• ESI-: Heater Temp 300°C; Sheath Gas Flow rate, 45 arb; Aux Gas Flow Rate, 15 arb; spray voltage, 3.2KV; Capillary Temp, 350°C; S-Lens RF Level, 60%.

• Data Analysis

The raw data was acquired and aligned using Compound Discover 3.1 software. After normalizing to total peak intensity, metabolites from both ESI- and ESI+ were merged and imported into Metaboanalyst 5.0 for downstream analysis, including principal component analysis, hierarchical clustering analysis, and volcano plots. The KEGG pathway database was used to explore metabolomic pathways. Venn diagrams were created using the Venny 2.1 interactive tool. One-way ANOVA followed by Šídák multiple comparisons test was performed for comparing amino acid profiles using GraphPad Prism version 9.4.1.

Results

1. Principal Component Analysis (PCA)

Principal Component Analysis (PCA) was conducted to identify variations in the metabolomic profiles of the different treatment groups. The PCA results revealed distinct clustering patterns between the control and salt-stressed samples for both O. sativa and O. coarctata. This separation indicates significant metabolic alterations in response to salt stress.

2. Differentially Accumulated Metabolites (DAMs)

Volcano plots were used to visualize differentially accumulated metabolites (DAMs) between control and salt-stressed conditions. The analysis identified several metabolites with significant fold changes in both species. For O. sativa, metabolites such as proline, betaine, and several organic acids showed significant increases under salt stress. In contrast, O. coarctata exhibited increases in metabolites like sucrose, trehalose, and specific amino acids, indicating distinct metabolic adjustments to counteract salt stress.

3. Hierarchical Clustering Analysis

Hierarchical clustering analysis further confirmed the distinct metabolic responses of the two species to salt stress. The heatmap demonstrated clear clustering of samples according to their treatment groups, with notable differences in the abundance of specific metabolites between O. sativa and O. coarctata. Metabolites involved in stress responses, energy metabolism, and osmotic regulation were particularly prominent.

(a) Score plot from PCA analysis of metabolite profiles of Oryza sativa and Oryza coarctata without and with salt stress samples. Red (left) and green (right) ellipsoids show a 95% confidence interval in Oryza coarctata without stress and with stress plants, respectively. (b) Hierarchical clustering for the top 500 metabolites of Oryza sativa and Oryza coarctata without and with salt stress samples.

4. KEGG Pathway Analysis

KEGG pathway analysis provided insights into the metabolic pathways affected by salt stress. Key pathways, including amino acid biosynthesis, carbohydrate metabolism, and lipid metabolism, were significantly enriched in response to salt stress in both species. However, the specific pathways and metabolites involved differed between O. sativa and O. coarctata, reflecting their varying tolerance mechanisms.

5. Targeted Metabolite Quantification

Targeted metabolomic analysis quantified specific metabolites, such as amino acids, organic acids, and lipids. Under salt stress, O. sativa showed increased levels of proline and glutamine, metabolites known for their roles in osmoprotection and stress responses. Conversely, O. coarctata demonstrated elevated levels of trehalose and sucrose, which are crucial for osmotic balance and energy storage.

6. Amino Acid Profile Analysis

One-way ANOVA followed by Šídák multiple comparisons test was performed to compare amino acid profiles. The results indicated significant differences in amino acid accumulation between the control and salt-stressed conditions in both species. Notably, O. sativa exhibited substantial increases in amino acids like proline, while O. coarctata showed significant changes in amino acids such as valine and leucine, suggesting species-specific adaptations to salt stress.

Grouped bar plot indicating enriched metabolite sets analysis in four comparison groups: Oc.C/Os.C, Oc.S/Oc.C, Os.S/Os.C, and Oc.S/Os.S. The number of differentially accumulated metabolites (|fold change|>1.5 and p < .05) that enrich a particular super-class of metabolite sets based on chemical structure for each group accounts for the height of each bar.

Volcano plots representing significantly modulated metabolites in comparison groups a) Oc.C/Os.C b) Oc.S/Oc.C c) Os.S/Os.C and d) Oc.S/Os.S [analysis cut-off: |fold change|>1.5 and p < 0.05] [red = upregulated; blue = downregulated; grey = nonsignificant].

Heatmap analysis depicting the logarithm of fold change values for lipids in four comparison groups: Oc.C/Os.C, Oc.S/Oc.C, Os.S/Os.C and Oc.S/Os.S. Only lipids that showed differential expression (|Fold change|>1.5 and p <.05) in at least one of the four comparison groups were included. Heatmap cells with |Fold change|≤1.5 are shown in grey, indicating no significant change.

Reference

1. Tamanna, Nishat, et al. "Comparative metabolite profiling of salt sensitive Oryza sativa and the halophytic wild rice Oryza coarctata under salt stress." Plant‐Environment Interactions 5.3 (2024): e10155.