Journal of Chromatography B

Volume 1185, 15 November 2021, 122987

Quantification of lectin in soybeans and soy products by

liquid chromatography-tandem mass spectrometry
Yang Wen a, Anguo Liu a, Chengzhen Meng a, Zhen Li b, Pingli He a

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https://doi.org/10.1016/j.jchromb.2021.122987
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Highlights

• The lectin in soybeans can be accurately quantified through detection of unique

reporter peptides.

• The detect limit of lectin was 35.5 μg/g.

• It provids a method for the evaluation of soy products with different soybean
processing techniques.

Abstract

Lectin is one of the major anti-nutritional factors in soybeans and inhibits digestion of dietary protein. Here, an
absolute quantification method was developed to detect lectin using synthetic peptide 183TTSWDLANNK192 as
reference standard and corresponding isotope labeled peptide TTSWDLANNK (Alanine-13C3,15N) as internal standard to
normalize results. After the ground soybeans and soy products were defatted with n-hexane and extracted with
extraction buffer, the crude protein extract was digested on filter membrane by trypsin. Further, the enzymatic
hydrolysis peptides were quantified using liquid chromatography-tandem mass spectrometry. The synthetic reference
peptide showed a detection limit of 0.27 ng/mL and a linear relationship in the range of 3.2–1000 ng/mL (r2 > 0.997).
Correspondingly, the detect limit of lectin in soybean samples was 35.5 μg/g. The results showed that the recoveries of
the lectin in spiked samples ranged from 80.9% to 108.7% with intra-day precisions (% CV) less than 9%. The method
was successfully used to evaluate lectin levels in hundreds of soybean seeds from different varieties and soy products
from different soybean processing techniques. Furthermore, the method may provide a potential application as a
general method for the ultrasensitive detection of various protein anti-nutritional factors in food.

Introduction

Soybean (Glycine Max) is an important food crop whose seeds are rich in oil and protein that is widely used in
pharmaceuticals and food [1]. Since soybeans contain about 40% protein and 20% fat, after edible oil is extracted from
soybeans, the remaining byproduct (soybean meal) is a high quality protein feed for livestock such as chickens, pigs
and dairy and beef cattle. However, there are some antinutritional factors in soybean which affect its utilization in
animal feeds, such as interfering with the digestion and absorption of nutrients, disturbing normal metabolism and
causing adverse physiological responses, and resulting in hypersensitivity and death [2], [3].

Lectin (also known as hemagglutinin or agglutinin) is a carbohydrate-binding protein that makes up 10% of total
soybean protein. Soybean lectin is a glycoprotein with a molecular weight of 11–120 kDa made up of four identical
subunits with two saccharide binding sites. It is used as a therapeutic drug that preferentially binds to the membranes
of cancer cell or their receptors to induce cytotoxicity and inhibit tumor growth [4]. Nevertheless, lectin is also a kind
of anti-nutritional factor present in the soybeans [5], [6] and accounts for approximately 50% of the growth inhibition
in rats fed unheated soybeans [7]. It can bind to the epithelium of the small intestine, which results in changes in the
morphology and function of the small intestine [8]. Moreover, lectin may enter the systemic circulation through
intestinal epithelial cells and can cause growth inhibition, organ damage and even death [9]. Therefore, lectin has been
demonstrated as a major anti-nutritional factor in soybeans. In fact, soybean breeders are developing new varieties
with low lectin content by selective breeding or some other gene targeting technology. In addition, commercialization
of soy products for human consumption and animal feeding needs to control lectin levels due to its anti-nutritional
effect.

The research shows that lectin is heat-labile and its activity can be reduced or eliminated by thermal treatment [10].
Recently, some technologies, such as expansion or fermentation, have been applied to reduce the content of active
lectin in soybeans [7], [11]. Therefore, there is an urgent need to provide sensitive and accurate methods for the
identification and quantification of lectin in soybean seeds and soybean products in order to identify potential hazards
and evaluate the effectiveness of breeding and processing techniques.

Due to the large molecular weight of lectin protein and its low content in soybean, there have been few reports on
sensitive and accurate detection of lectin in soybean and its processed products. Initially, lectin has been indirectly
determined by a time-consuming and cumbersome hemagglutination assay using red blood cell [12], more recently, by
enzyme-linked immunosorbent assay (ELISA) [13]. However, the immunoassays may be subject to other limitations
such as antibody availability, lower specificity and sensitivity. Due to the presence of many allergen proteins with
similar structure in soybean, the antibodies may cross-react with other allergens, resulting in reduced specificity and
sensitivity of the method. Therefore, immunoassay is commonly applied as a semi-quantitative method. Liquid
chromatography with UV detection or tandem mass spectrometry has used been as a powerful method for the
discovery and quantitative detection of peptides and proteins in complex matrices containing a variety of proteins [14],
[15], [16]. For example, Anta et al. reported an analytical method for the determination of lectin in soybean crops using
perfusion high-performance liquid chromatography [17]. Because soybeans are rich in proteins, HPLC did not
effectively separate the individual proteins, which lead to low sensitivity and selectivity.

The protein absolute quantification (AQUA) technology based on liquid chromatography with tandem mass
spectrometry (LC–MS/MS) is routionely performed to quantify proteins in samples using synthetic internal or external
standard peptide [15], [18], [19], [20]. Compared with ELISA, this technique results in accurate and sensitive
quantification of a target protein without the need for a well-characterized antibody and highly purified protein
standard. For example, Hill et al. reported a quantitative multiplex analysis of 10 potential allergens in soybean seeds
using synthetic isotope-labeled peptides as internal standards [21]. Similarly, our group has reported a quantitative
analytical method using AQUA to measure Gly m 5.0101 in soybeans and soy products [22]. Therefore, the objective of
the present study was to develop an AQUA assay for the quantitative determination of lectin in soybeans and soy
products. The sensitivity, linearity, recovery and accuracy of the method were evaluated. The presence of lectin traces
in the soy products were clearly discernible through detection of unique reporter peptides. To the best of our
knowledge, this study represents the first absolute quantitative assay for an endogenous lectin in soybean meals.
Therefore, this study will provide strong technical support for evaluating the effects of different soybean processing
technologies.

Section snippets
Reagents and chemicals

Trypsin (Mass Spectrometry Grade) was obtained from Promega (Madison, WI). Lectin protein standard,
iodoacetamide, dithiothreitol and ammonium bicarbonate were obtained from Sigma Company (St. Louis, MO). BCA™
Protein Assay Kit was obtained from Pierce (Rockford, IL). Soybean lectin ELISA kit was obtained from Dogesce (Beijing,
China). HPLC grade acetonitrile and formic acid were obtained from Fisher Scientific International (Hampton, NH).
10 kDa MWCO ultrafiltration filters ware purchased from …

Selection of quantitation peptide

For the AQUA technology, the selection of quantitation peptide was a key challenge during assay development. In
general, peptide used for absolute quantification should satisfy the following principles: the peptide should be a highly
species-specific and conserved sequence of 9–20 amino acids; the peptide should not contain cysteine, histidine,
methionine and other unstable amino acids; the peptide segment does not contain continuous lysine or arginine and
post-translational modification and…

Discussion

In recent years, a variety of processing methods have been developed to reduce soy antigenic protein. For example, it
has been reported that lectin could be inactivated by wet heat or biochemical treatments [24], [25]. For wet expansion,
high temperature, high pressure and high shear force destroyed the structure of soybean protein and reduced the
sensitization of soybean. Li et al. found that lectin could be completely inactivated at 120 °C [26]. A 15 min wet heating
was sufficient to reduce…

Conclusion

A LC-MS/MS method was developed for the determination of soybean lectin by using the synthetic external standard
peptide as quantitative peptide. The peak of the characteristic peptide TTSWDLANNK was clearly visible in MRM
chromatography, and it was well distinguished from other coexisting peptides in soybean protein extract. The method
showed an acceptable sensitivity with a LOD at 35.5 μg/g, recoveries with 80.9–108.7%, and intra-day coefficient of
variation less than 9%. The results of…

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.…

Acknowledgements
The National Natural Science Foundation of China (31972602) is gratefully acknowledged.…

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