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Challenges
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ELISA kits oftentimes are used by technicians who are not experienced
at LC/MS/MS.
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Allergens are present in many matrices.
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Method development to determine allergen proteins is time
consuming.
•
Most food testing labs are used to using LC/MS/MS for small molecule
contaminant testing.
•
Sample preparation for allergens (protein digestion) is significantly
different from current sample prep methods (LLE, QuEChERS, SPE, etc)
Challenges
•
Unique protein and peptide selections with different digestion conditions
Perform protein ID for allergens: Trypsin; chymotrypsin; species selected based on
–
the list highlighted by The Codex Alimentarius.
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Search against database and select unique proteins/peptides.
Goal
:
•
Detection of two proteins each species (if available), 3‐4 peptides each protein, and 3‐4
MRM transitions each peptide.
•
MRM transition selections with consideration on sensitivity and few/no interferences
from the food matrix.
•
MRM transition selections made with consideration of potential modification during
food processing (cooking, baking, fermenting etc. )and storage (i.e. MRMs must be
detectable in raw and processed food)
•
Sample clean up: lipids removal
–
Try different solvents (ex. hexane, etc)
•
Protein extraction and digestion optimization
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Try different denature conditions: (ex. heat, urea, OGS, guanidine).
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Final combined mixture of denature reagent.
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Must be simple for sample preparation and easy to reproduce.