Use of specific peptide biomarkers for quantitative confirmation of hidden allergenic peanut proteins Ara h 2 and Ara h 3/4 for food control by liquid chromatography–tandem mass spectrometry

Abstract

A liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS-MS) method based on the detection of biomarker peptides from allergenic proteins was devised for confirming and quantifying peanut allergens in foods. Peptides obtained from tryptic digestion of Ara h 2 and Ara h 3/4 proteins were identified and characterized by LC-MS and LC-MS-MS with a quadrupole-time of flight mass analyzer. Four peptides were chosen and investigated as biomarkers taking into account their selectivity, the absence of missed cleavages, the uniform distribution in the Ara h 2 and Ara h 3/4 protein isoforms together with their spectral features under ESI-MS-MS conditions, and good repeatability of LC retention time. Because of the different expression levels, the selection of two different allergenic proteins was proved to be useful in the identification and univocal confirmation of the presence of peanuts in foodstuffs. Using rice crisp and chocolate-based snacks as model food matrix, an LC-MS-MS method with triple quadrupole mass analyzer allowed good detection limits to be obtained for Ara h 2 (5 microg protein g(-1) matrix) and Ara h 3/4 (1 microg protein g(-1) matrix). Linearity of the method was established in the 10-200 microg g(-1) range of peanut proteins in the food matrix investigated. Method selectivity was demonstrated by analyzing tree nuts (almonds, pecan nuts, hazelnuts, walnuts) and food ingredients such as milk, soy beans, chocolate, cornflakes, and rice crisp.

Primary sequence and site‐selective hydroxylation of prolines in isoforms of a major peanut allergen protein Ara h 2

“…Previous characterization of Ara h 2 provided the identification of an N-terminal peptide [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] and two tryptic peptides [82][83][84][85][86][87][88][89][90][91][92][93][94] and [95][96][97][98][99][100][101][102][103][104][105][106][107][108][109][110], which were proposed as potential biomarkers for regulatory monitoring. 6 A recent study of 2-D gel electrophoretic patterns of proteins from the Virginia type peanut identified fourteen spots that were linked to Ara h 2 by partial peptide mass fingerprinting. 7 In a separate study in 1992 focused on potential post-translational modifications (PTM), Burks et al proposed that Ara h 2 is a glycoprotein, based on periodic acid-Schiff staining.…”

Allergenicity of Milk Proteins

“…SRM is presently considered the gold standard for absolute quantification, whereas multiple reaction monitoring (MRM) can monitor the masses of selected signature peptides. For an allergen evaluation, internal reference peptides are required for food product monitoring by LC-SRM MS [124]. Hydrolyzed protein samples are spiked with known amounts of synthetic peptides and monitored by LC-MS in the SRM mode.…”

Food Allergen Analysis: Detection, Quantification and Validation by Mass Spectrometry

“…Table 4a. Quantification of food allergens in different food products by mass spectrometry using label-free quantification with an (1) external calibration curve [75][76][77][78][79]88], (2) unlabelled modified synthetic peptide [78], and (3) standard addition [79]. Table 4b.…”

“…Using a calibration curve obtained by spiking fruit juice with extracted milk proteins, they found recoveries between 68 and 79% [75]. This strategy was also used to quantify peanut proteins in rice crispy/chocolate snacks [76]. A significant suppression effect, ranging from 30 to 50%, was observed for the Ara h2 peptide signal, while suppression of the Ara h3/4 peptide signal was less than 10%.…”

“…Published methods, however, do not always meet the AOAC requirements, even with spiked samples. For instance, Careri et al [76] observed a suppression effect between 30 and 50% for the Ara h2 peptide signal, and Monaci et al [75] obtained recoveries ranging from 68 to 79% for α-lactalbumin and β lactoglobulin. Altogether, these works show that internal standards are needed for the quantification of allergens in food matrices.…”