Amylopectin Chain Length Distribution Profiling

What is Amylopectin?

Amylopectin, the counterpart of amylose, is a polysaccharide containing a branched structure with mostly short chains of α-(1→4)-linked anhydroglucose units which are interlinked by α-(1→6)-glucosidic linkages at approximately every 20 to 30 anhydroglucose units. It is made up of around 1000 glucose units and is one of the largest molecules found in nature. It is one of the two main components in starches.

Amylopectin is a highly-branched molecule, and those branch points in Amylopectin make up approximately 4 to 6% of total linkages. It consists of three different types of chains. The A chains, also known as the unbranched chains, are the linear segments linked to other B or C chains by their reducing ends through a single α-(1→6)-linkage. The B chains are those linked to other B or C chains through α-(1→6)-linkages and also carry one or more A or B chains attached to them. The C chains are the single, central chain that carries the only reducing group of the amylopectin molecule. Each amylopectin molecule has only one C chain.

A number of parameters have been used in order to describe the structural characteristics of amylopectin molecules from various botanical sources. As one of the key parameters, amylopectin chain length distribution is the first structural level and involves individual chains from a branch point. It plays a very important role to determine gelatinization temperature and to characterize the structural of amylopectin.

Figure 1. Examples of amylopectin branched-chain length distribution of various types of starches. The average branch chain length of amylopectin has a bimodal distribution and each starch has a distinct branch chain length distribution profile.

Glycomics and Amylopectin Analysis at Creative Proteomics

Glycomics is the comprehensive study of glycan structures within biological molecules, and it plays a crucial role in understanding the functional properties of polysaccharides like amylopectin. At Creative Proteomics, we provide advanced glycomics analysis services to explore the glycosylation patterns in amylopectin. By leveraging state-of-the-art technologies, we can offer detailed insights into the branching structures and glycan distributions, which are essential for characterizing the molecular and functional properties of amylopectin.

Technology Platforms for Amylopectin Chain Length Distribution Profiling

Creative Proteomics utilizes High-Performance Anion-Exchange Chromatography (HPAEC), a technique that separates and quantifies the different chain lengths of amylopectin by exploiting their varying degrees of polymerization. This method includes deproteinization of starch with protease and sodium bisulfite, followed by hydrolysis and debranching with isoamylase, and final analysis using HPAEC. The chain length distribution is generally characterized as a percentage of the total peak area. The branch chains of amylopectin are fractionated and defined as A chains (DP 6–12), B1 chains (DP 13–24), B2 chains (DP 25–36), and B3 chains (DP > 37). This technique offers high resolution and sensitivity, making it possible to detect minor variations in chain length distribution.

We also employ Size-Exclusion Chromatography (SEC), which separates amylopectin chains based on their molecular size, providing a comprehensive profile of the chain length distribution. This technique involves the preparation and fractionation of amylopectin molecules, enabling the separation based on molecular size to deliver an accurate profile of the chain length distribution. SEC is capable of analyzing a wide range of molecular sizes, delivering a complete overview of the amylopectin structure.

Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) is another technique we use. It involves fluorophore labeling and electrophoresis to analyze the distribution of amylopectin chains. Samples are labeled with fluorophores and subjected to electrophoresis, allowing for the detection and quantification of various chain lengths. This method is highly sensitive and accurate, suitable for detecting low-abundance chain lengths.

For detailed molecular weight analysis, we utilize MALDI-TOF Mass Spectrometry. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) is used to determine the molecular weights of amylopectin chains. The amylopectin samples are ionized and analyzed to determine the molecular weights of the chains, providing high-resolution mass spectrometric data that allows precise determination of chain lengths.

Enzymatic Digestion and Analysis is a technique that involves the enzymatic digestion of amylopectin followed by chromatographic or electrophoretic analysis. Amylopectin samples are digested with specific enzymes to break down the chains, followed by analysis to profile the resulting fragments. This method offers detailed insights into the branching structure and chain length distribution, enhancing the understanding of amylopectin functionality.

Table 1. Branch chain length distributions of amylopectins

Sample Requirements

To ensure the accuracy and reliability of our amylopectin chain length distribution profiling services, we have specific requirements for sample types and quantities. The following table outlines the recommended sample types and volumes:

Sample Preparation Guidelines

• Plant Tissue: Fresh or frozen samples should be provided, avoiding exposure to extreme temperatures or light.
• Starch Extract: Ensure the extract is free from contaminants and properly labeled.
• Processed Food Products: Homogenize the samples thoroughly to ensure uniformity.
• Purified Amylopectin: Samples should be lyophilized or in solution with appropriate preservatives.

Deliverables

Creative Proteomics is committed to delivering comprehensive and detailed reports for amylopectin chain length distribution analysis. Our analytical reports typically include:

Chain Length Distribution Profiles

• Graphical representations of chain length distributions, highlighting major and minor peaks.
• Quantitative data on the proportion of different chain lengths within the sample.

Molecular Weight Analysis

• Detailed molecular weight distribution data obtained from techniques like MALDI-TOF MS.

Branching Structure Insights

• Analysis of branching patterns and their impact on amylopectin functionality.

Methodology and Technical Notes

• Comprehensive documentation of the analytical methods used.
• Technical notes on sample preparation, analysis conditions, and data interpretation.

Recommendations and Conclusions

• Expert interpretation of the data, providing insights into the implications of the chain length distribution.
• Recommendations for further analysis or potential applications based on the findings.

As one of the leading companies in the omics field with over years of experience in omics study, Creative Proteomics provides glycomics analysis service customized to your needs. Contact us to discuss your project.

Amylopectin chain length distribution in grains of japonica rice as affected by nitrogen fertilizer and genotype

Journal: Journal of Cereal Science
Published: 2016

Background

Nitrogen fertilization plays a crucial role in determining the quality of rice grains. Different genotypes of rice respond variably to nitrogen treatments, affecting grain appearance, chemical composition, and various physicochemical properties. Understanding these effects can help optimize fertilization strategies to improve rice yield and quality.

Technical Methods

This study employed a variety of technical methods to analyze the effects of nitrogen fertilization on the grain quality and amylopectin structure in japonica rice. The methods included:

Nitrogen Treatments

Different rates and timings of nitrogen (N) fertilization were applied to the rice plants. These treatments included both low and high N rates, as well as specific timings such as spikelet-sustaining fertilization. The objective was to observe how varying N levels and application schedules impacted the rice grain's appearance, chemical composition, and physicochemical properties.

Grain Appearance and Composition Analysis

The grain appearance and composition were thoroughly analyzed using various measurements, including:

• Grain weight
• Length/width ratio
• Chalky rice ratio
• Starch content
• Amylose content
• Protein content
• Crude fat content

These analyses provided insights into how N fertilization affected the overall quality and nutritional value of the rice grains.

Amylopectin Chain Length Distribution

High-performance size-exclusion chromatography (HPSEC) was utilized to determine the amylopectin chain length distribution. This method separates the amylopectin chains based on their size, allowing for a detailed profile of the chain lengths. The glycomics approach was integrated to examine the glycan structures within the amylopectin, providing a comprehensive understanding of the glycosylation patterns and their impact on the rice starch properties. This integration of glycomics enabled the identification and quantification of various glycan structures, crucial for understanding the functional properties of amylopectin.

Starch Pasting Properties

A Rapid Visco Analyzer (RVA) was employed to measure the pasting properties of the rice starch. The parameters analyzed included:

• Peak viscosity (PKV)
• Hot paste viscosity (HPV)
• Breakdown viscosity (BDV)
• Cool paste viscosity (CPV)
• Setback viscosity (SBV)
• Pasting temperature (PaT)

These measurements provided insights into the starch's behavior during heating and cooling, which is essential for understanding its cooking and processing characteristics.

Gelatinization and Retrogradation

Differential scanning calorimetry (DSC) was used to analyze the gelatinization and retrogradation properties of the starch. This technique measures the heat flow associated with the starch's phase transitions, providing detailed information on:

• Gelatinization temperature
• Gelatinization enthalpy
• Retrogradation temperature
• Retrogradation enthalpy

These parameters are critical for understanding how the starch behaves during cooking and storage.

Crystalline Structure

The relative crystallinity (RC) of the starch was calculated to understand its crystalline structure. X-ray diffraction (XRD) was employed to obtain the diffraction patterns, which were then analyzed to determine the RC values. This analysis provided insights into the molecular arrangement of the starch granules and how N fertilization affected their crystallinity.

Results

• Grain Appearance and Composition: N fertilization significantly affected grain appearance and composition. High N rates slightly decreased starch and amylose contents in Wuyujing3 but increased total protein and decreased crude fat in Wuyunjing7.
• Amylopectin Chain Length Distribution: High N rates reduced the percentage of short amylopectin chains and the ratio of short to long chains in both cultivars. The timing of N application also affected the chain length distribution.
• Starch Pasting Properties: N fertilization generally lowered PKV, HPV, BDV, and CPV values. Wuyujing3 exhibited a softer texture with lower CPV and SBV compared to Wuyunjing7.
• Gelatinization and Retrogradation: High N rates increased gelatinization temperatures and decreased gelatinization enthalpy. No significant influence on retrogradation parameters was found, although Wuyujing3 showed lower retrogradation enthalpy compared to Wuyunjing7.
• Crystalline Structure: N treatments did not significantly affect the relative crystallinity of the starch. Wuyujing3 had lower RC values than Wuyunjing7, possibly due to differences in amylopectin structure.

Representative distribution of rice debranched amylopectin. a) HPSEC-normalized number distribution variance, b) Starch size-weight distribution, c) Chain length number distribution, d) Model fitting of amylopectin biosynthesis.

Reference

1. Yang, Xiaoyu, et al. "Amylopectin chain length distribution in grains of japonica rice as affected by nitrogen fertilizer and genotype." Journal of cereal science 71 (2016): 230-238.