Feasibility of Utilizing Stable-Isotope Dimethyl Labeling in Liquid Chromatography⁻Tandem Mass Spectrometry-Based Determination for Food Allergens-Case of Kiwifruit

Abstract

Stable-isotope dimethyl labeling is a highly reactive and cost-effective derivatization procedure that could be utilized in proteomics analysis. In this study, a liquid chromatography- tandem mass spectrometry in multiple reaction monitoring mode (LC-MS-MRM) platform for the quantification of kiwi allergens was first developed using this strategy. Three signature peptides for target allergens Act d 1, Act d 5, and Act d 11 were determined and were derivatized with normal and deuterated formaldehyde as external calibrants and internal standards, respectively. The results showed that sample preparation with the phenol method provided comprehensive protein populations. Recoveries at four different levels ranging from 72.5-109.3% were achieved for the H-labeled signature peptides of Act d 1 (SPA1-H) and Act d 5 (SPA5-H) with precision ranging from 1.86-9.92%. The limit of quantification (LOQ) was set at 8 pg mL^-1 for SPA1-H and at 8 ng mL^-1 for SPA5-H. The developed procedure was utilized to analyze seven kinds of hand-made kiwi foods containing 0.0175-0.0515 mg g^-1 of Act d 1 and 0.0252-0.0556 mg g^-1 of Act d 5. This study extended the applicability of stable-isotope dimethyl labeling to the economical and precise determination of food allergens and peptides.

Keywords: food allergen; kiwifruit; liquid chromatography–tandem mass spectrometry; stable-isotope dimethyl labeling.

Figure 1

Reaction and m/z shift of (i) light (hydrogen) and (ii) heavy (deuterium) stable-isotope dimethyl labeling. R represents the remainder of the peptide and M represents the m/z of the native peptide with a single charge.

Figure 2

SDS-PAGE of the kiwifruit extracts from four different protein extraction methods. Lane 1, protein ladder; lane 2, phenol method; lane 3, ammonium sulfate method; lanes 4 and 5, TCA method; lanes 6 and 7, sodium chloride method. Arrow indicators on the right indicate four indicator proteins, glycoprotein (Act d 3) (40 kDa), actinidin (Act d 1) (30 kDa), kiwellin (Act d 5) (26 kDa), TLP (thaumatin-like protein) (Act d 2) (24 kDa).

Figure 3

Protein sequences of (A) actinidin (Act d 1), (B) kiwellin (Act d 5), and (C) kirola (Act d 11). The tryptic peptides identified with LC/ESI-Q-ToF are underlined, and the selected signature peptides are presented in boldface type.

Figure 4

Mass spectra of (A) SPA1-H and (B) SPA1-D. The dotted line represents the m/z of SPA1 and Δm/z shows the m/z shift of the signature peptide before and after stable-isotope dimethyl labeling.

Figure 5

MRM chromatograms of (A) SPA1-H (1 μg mL⁻¹) and (B) SPA1-D (500 ng mL⁻¹) for the mixture of peptide standards and (C) SPA1-H (479.3 ng mL⁻¹) and (D) SPA1-D (500 ng mL⁻¹) for kiwifruit raw extract samples.

Figure 6

Content of (A) Act d 1 and (B) Act d 5 in one gram of kiwi foods produced by different processing. There was no significant difference between treatments with the same letter (p-value < 0.05. n = 3).