Protein characterization: LDP 2020-2021 Reference Method

Overview
Objectives

  • To evaluate variation of protein and amino acid compositions in cultivated lentils from Canada using the Dumas Combustion method.

  • Compare this reference wet chemistry method to determine protein and amino acid content with NIRS methods done in a related experiment.

Parent Research Study
Characterize protein levels and composition in Lentil germplasm to inform breeding targets
Experiment Summary

Protein contents were determined by the Dumas Combustion method using AOAC Official Method 990.03. The conversion factor was set as 6.25 to convert percent nitrogen to percent crude protein (N × 6.25). The percent dry matter was analyzed according to AOAC Official Method 930.15.

All amino acids, except Met, Cys and Trp, were analyzed by the regular acid hydrolysis using AOAC Official Method 982.30. Basically, these amino acids were determined in three steps: acid hydrolysis, derivation and UHPLC separation and detection. Two sulphur amino acids (i.e., Met and Cys) were determined according to AOAC Official Method 985.28, where samples were first oxidized with performic acid followed by acid hydrolysis. The Trp content was analyzed by alkaline hydrolysis, which was positioned by the International Organizations for Standardization 13904:2005. For amino acid analysis, the free amino acid molecular weights were used to calculate amino acid contents.

 

Germplasm
Germplasm Genus
Lens
Germplasm Scientific Name
Lens culinaris
Germplasm Collection
AGILE Lentil Diversity Panel
Phenomics
Key Measurements
  • Amino Acid specific percent composition on a dry matter basis (%) for each of 18 amino acids
  • Total protein percent composition on a dry matter basis (%)
Experimental Design
Experimental Site Locations
  • Sutherland, Saskatchewan, Canada
  • Rosthern, Saskatchewan, Canada
Timepoints
  • 2016
  • 2017
Biochemical Technique
  • Dumas Combustion method (Official methods of analysis of AOAC International, n.d.)
  • Nitrogen Analysis (Central Testing Labs; Winnipeg, MB, Canada)
Experimental Design

Protein contents were determined by the Dumas Combustion method using AOAC Official Method 990.03. The conversion factor was set as 6.25 to convert percent nitrogen to percent crude protein (N × 6.25). The percent dry matter was analyzed according to AOAC Official Method 930.15. All amino acids, except Met, Cys and Trp, were analyzed by the regular acid hydrolysis using AOAC Official Method 982.30. Basically, these amino acids were determined in three steps: acid hydrolysis, derivation and UHPLC separation and detection. Two sulphur amino acids (i.e., Met and Cys) were determined according to AOAC Official Method 985.28, where samples were first oxidized with performic acid followed by acid hydrolysis. The Trp content was analyzed by alkaline hydrolysis, which was positioned by the International Organizations for Standardization 13904:2005. For amino acid analysis, the free amino acid molecular weights were used to calculate amino acid contents.

Attribution
Researchers
Data Collector
Jiayi Hang
Data Custodian
Data Curator
Ruobin Liu
Publication(s)
Associated Datasets

LDP Protein Percent Composition Reference Dataset (XLSX, TSV)

This dataset consists of the total protein percent composition on a dry matter basis and amino acid specific percent composition on a dry matter basis (%) for each of 18 amino acids. The protein compositions were determined using the Dumas Combustion method and this dataset will act as a reference to evaluate the validity of the NIR results in a linked experiment. The seeds used for this analysis are from the AGILE field trial site-years Rosthern2016, Sutherland2016, Rosthern2017, Sutherland2017.

Funding
Research Organization
Funding Grant
EVOLVES: Enhancing the Value of Lentil Variation for Ecosystem Survival
  • Kirstin E Bett
  • Albert Vandenberg
University of Saskatchewan

2019-2023