Quantification of Allergenic Bovine Milk α_S1-Casein in Baked Goods Using an Intact ¹⁵N-Labeled Protein Internal Standard

Abstract

Intact bovine ¹⁵N-α(S1)-casein was used as an internal standard in a selected reaction monitoring (SRM) assay for milk protein in baked food samples containing fats, sugar, and gums. Effects on SRM results of sample matrix composition in two biscuit recipes containing nonfat dry milk (NFDM) were studied, including samples from a milk allergen ELISA proficiency trial. Following extraction of defatted samples with carbohydrate-degrading enzymes and acid precipitation of casein, the SRM assay exhibited an LOQ of <3 ppm NFDM with 60-80% recovery. NFDM levels measured by the SRM assay were 1.7-2.5 times greater than median levels determined by ELISA. Differences were observed in the α(S1)-casein interpeptide SRM ion abundance profile between recipes and after baking. ¹⁵N-α(S1)-Casein increases SRM analysis accuracy by correcting for extraction recovery but does not eliminate underestimation of allergen concentrations due to baking-related milk protein transformation (modifications).

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

“…Table 4b. Quantification of food allergens in different food products by mass spectrometry using stable isotope labelling quantification with an (1) isotope-labelled protein [82], (2) isotope-labelled peptide [59,61,71,75,84,89,90] or (3) long isotope-labelled peptide [83].…”

“…Although more expensive than the strategies described above, this approach has the advantage that the unlabelled and isotope-labelled peptides show similar ionization and similar mass spectrometry response signals. For allergen quantification, three kinds of isotope-labelled standards exist [81]: proteins [82], concatemers [83] (or long isotope-labelled peptides) and Aqua peptides [61,71,75,84] (isotope-labelled peptides) (Figure 5). …”

“…Newsome et al studied the recovery of the milk allergen α-S1 casein in baked cookies using a labelled internal α-S1 casein, and obtained recoveries ranging from 60 to 80% [82]. Use of an internal standard allows correcting for the matrix effect and for effects linked to different steps in the sample preparation protocol (protein extraction and enzymatic digestion).…”

Detection of Allergen Markers in Food: Analytical Methods

“…A simplifi ed extraction containing fi ammonium bicarbonate and urea was validated in protein-rich infant cereals without additional enrichment or solid-phase purifi cation. Optimizing fi the detection of αS1-casein, allergen peptides derived from milk-incurred cookie samples were quantitated using 13 C 15 N-labeled peptide standards and a stable isotope-labeled protein [183]. Isotope-labeled 15 N-αS1-casein improved SRM analysis with regards to extraction recovery; however, it did not eliminate the underestimation of allergen concentration arising from thermal processing during baking.…”

“…Isotope-labeled 15 N-αS1-casein improved SRM analysis with regards to extraction recovery; however, it did not eliminate the underestimation of allergen concentration arising from thermal processing during baking. Extraction conditions were optimized for the detection of casein in allergen-incurred cookie samples with an LOQ < 3 ppm of nonfat dry milk and an estimated recovery between 60-80% [183]. Alternatively, Zhang et al [184] designed a peptide construct for α-lactalbumin with flanking amino acid sequences at the C-and fl N-termini.…”