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

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 mu M for most amino acids (mean 0.017 mu M).

Automated summary

A fast LC-MS method for determining amino acids has been developed, requiring only three simple steps of hydrolysis, neutralisation, and sample dilution with a borate buffer solution. The method shows excellent repeatability and reproducibility for amino acid content, peak area, and retention time. Detection levels on column are below 0.1 μM for most amino acids.