Advancing Targeted Food Allergen Detection: Extraction Optimization and Sample Preparation Method Comparison Using LC-MS/MS

Authors

• Zhenhao Wang, University of Nebraska-LincolnFollow

First Advisor

Philip E. Johnson

Second Advisor

Melanie L. Downs

Committee Members

Joseph L. Baumert

Date of this Version

5-2026

Document Type

Thesis

Citation

A thesis presented to the faculty of the Graduate College at the University of Nebraska in partial fulfillment of requirements for the degree of Master of Science

Major: Food Science and Technology

Under the supervision of Professors Philip E. Johnson and Melanie L. Downs

Lincoln, Nebraska, May 2026

Comments

Copyright 2026, Zhenhao Wang. Used by permission

Abstract

Accurate quantification of food allergens in processed foods by liquid chromatography-tandem mass spectrometry (LC-MS/MS) requires effective protein extraction and sample cleanup. However, no single sample preparation workflow has shown consistent performance across diverse food matrices. This study evaluated extraction buffer formulations and post-extraction cleanup strategies for quantification of milk, soy, and peanut allergens in multiple processed food matrices.

Six extraction buffers with varying combinations of SDS, urea, and thiourea alongside a common Tris-HCl/DTT/PVPP base were evaluated across five food matrices. No significant differences in extraction efficiency were observed among buffer formulations for nonfat dry milk (NFDM), non-roasted soy flour, peanut flour, and cookies, suggesting that the inclusion of at least one denaturing agent is sufficient for effective protein extraction from these matrices. In dark chocolate, the SDS-containing buffer (1% SDS, 50 mM Tris-HCl, 20 mM DTT, 1% PVPP) achieved significantly higher protein recovery than all other formulations and was therefore selected for subsequent cleanup evaluation.

Three post-extraction cleanup workflows, including filter-aided sample preparation (FASP), single-pot solid-phase-enhanced sample preparation (SP3), and suspension trapping (S-Trap), were compared for SDS removal efficiency, peptide detection sensitivity, and quantitative recovery of six milk allergen marker peptides. All three workflows effectively removed SDS to LC-MS/MS-compatible levels. SP3 was excluded due to insufficient peptide recovery, attributable to incomplete on-bead tryptic digestion. Urea-based FASP was carried forward as the reference method over SDS-based FASP due to SDS loading limitations of the ultrafiltration membrane and compared against SDS-based S-Trap.

Calibration curves confirmed that both urea-FASP and S-Trap achieved detection sensitivity at or below 10 mg·kg^-1 NFDM. In incurred cookies, both workflows demonstrated comparable β-casein recovery with acceptable repeatability. In incurred dark chocolate, S-Trap yielded significantly higher β-casein recovery than FASP, though this difference may partly reflect the SDS-based extraction rather than cleanup method alone. FASP provided more consistent inter-day reproducibility across both matrices, supporting its reliability as a general-purpose workflow, while S-Trap represents a promising alternative for lipid-rich matrices.

Advisors: Philip E. Johnson and Melanie L. Downs