High-Throughput Analysis of Amino Acids for Protein Quantification in Plant and Animal-Derived Samples Using High Resolution Mass Spectrometry.

Priyanka Reddy,Joe Panozzo,Aaron Elkins,Simone J Rochfort

doi:10.3390/molecules26247578

Priyanka Reddy, Joe Panozzo + Show 2 more

Open Access

https://doi.org/10.3390/molecules26247578

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Abstract

Current methods for measuring the abundance of proteogenic amino acids in plants require derivatisation, extended run times, very sensitive pH adjustments of the protein hydrolysates, and the use of buffers in the chromatographic phases. Here, we describe a fast liquid chromatography–mass spectrometry (LC–MS) method for the determination of amino acids that requires only three steps: hydrolysis, neutralisation, and sample dilution with a borate buffer solution for pH and retention time stability. The method shows excellent repeatability (repeated consecutive injections) and reproducibility (repeated hydrolysis) in the amino acid content, peak area, and retention time for all the standard amino acids. The chromatographic run time is 20 min with a reproducibility and repeatability of <1% for the retention time and <11% for the peak area of the BSA and quality control (QC) lentil samples. The reproducibility of the total protein levels in the hydrolysis batches 1–4 was <12% for the BSA and the lentil samples. The level of detection on column was below 0.1 µM for most amino acids (mean 0.017 µM).

Highlights

The amino acid composition of pulses is an important factor contributing to the nutritional quality of proteins
We report for the first time extensive data on the repeatability of the hydrolysis methods and their injection, retention times, and peak area data that provides a robust and accurate measurement of amino acids using plant and animal-derived bovine serum albumin (BSA) samples
Formic acid) and acetonitrile (0.1% formic acid), and a linear gradient of 2% acetonitrile to 100% acetonitrile, which resulted in the chromatographic separation of the isobars, leucine (Leu), and isoleucine (ILeu), as well as amino acids such as asparagine and aspartic acid [9] that are one mass unit apart

Summary

Introduction

The amino acid composition of pulses is an important factor contributing to the nutritional quality of proteins. The assessment of amino acids can guide the selection of the germplasm within plant breeding programs; due to the current time-consuming sample preparation techniques, it is often not assessed. Protein percentage is often used as a surrogate for nutritional quality. The high levels of protein in pulses make it a healthy alternative to meat-derived protein. The environmental pressures on sustainable farming have spurred research activities in plant-based proteins and in improving the quality of the protein in pulses. To increase the appeal of pulses as an alternative protein source to incorporate into the human diet, the molecular phenotypes must be assessed so to underpin the genomic approaches to improve the breeding of pulses for improved human health

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