High-throughput analysis of amino acids in plant materials by single quadrupole mass spectrometry

Abstract

Background: The amino acid profile of plants is an important parameter in assessments of their growth potential, resource-use efficiency and/or quality as food and feed. Screening studies may involve large number of samples but the classical amino acid analysis is limited by the fact that it is very time consuming with typical chromatographic run times of 70 min or more.

Results: We have here developed a high-throughput method for analysis of amino acid profiles in plant materials. The method combines classical protein hydrolysis and derivatization with fast separation by UHPLC and detection by a single quadrupole (QDa) mass spectrometer. The chromatographic run time is reduced to 10 min and the precision, accuracy and sensitivity of the method are in line with other recent methods utilizing advanced and more expensive mass spectrometers. The sensitivity of the method is at least a factor 10 better than that of methods relying on detection by fluorescence or UV. It is possible to downscale sample size to 20 mg without compromising reproducibility, which makes the method ideal for analysis of very small sample amounts.

Conclusion: The developed method allows high-throughput analysis of amino acid profiles in plant materials. The analysis is robust and accurate as well as compatible with both free amino acids and protein hydrolysates. The QDa detector offers high sensitivity and accuracy, while at the same time being relatively simple to operate and cheap to purchase, thus significantly reducing the overall analytical costs compared to methods based on more advanced mass spectrometers.

Keywords: Amino acid analysis; Biomass; Biorefinery; Green leaves; Mass spectrometry; Protein extraction.

Fig. 1

Chromatogram of the standard amino acid solution showing an overlay of all mass channels. Differently coloured lines indicate different mass channels. All peaks had a corresponding stable isotope internal standard peak at the same retention time (not shown). The only exception was hydroxyproline for which no internal standard is available. Retention times of various amino acids are listed in Table 2

Fig. 2

Recoveries of amino acids measure in eight different biological materials. Box plot of measured amino acid concentrations of eight different sample materials relative to corresponding values obtained from an external certified laboratory or certified values of the reference material. Box edges denotes first and third quartile. Line in box denotes median value. Whiskers denote highest and lowest values

Fig. 3

Measured amino acid concentrations compared with corresponding certified values for NIST 1849a. A comparison of the measured values (n = 9) and the certified values of the reference material NIST 1849a. Error bars denote standard deviation

Fig. 4

Average relative standard deviation of amino acid concentrations (n = 5) analyzed in increasing quantities of two different plant matrixes, viz. spinach (shaded columns) and a protein concentrate of ryegrass (black columns). Different letters indicate significant statistical difference based on ANOVA (p ≤ 0.05)