Methods and systems for selective quantitation and detection of allergens including Gly m 7
View Patent ↗The invention relates to methods and systems taking advantage of bioinformatic investigations to identify candidate signature peptides for quantitative multiplex analysis of complex protein samples from plants, plant parts, and/or food products using mass spectroscopy. Provided are use and methods for selecting candidate signature peptides for quantitation using a bioinformatic approach. Also provided are systems comprising a chromatography and mass spectrometry for using selected signature peptides.
1. A method of selecting candidate signature peptide for quantitation of known allergen and potential allergens from a plant-based sample, wherein the potential allergens comprise at least one sequence selected from SEQ ID NOs: 13-15, comprising:
(a) identifying potential allergens based on homology to at least one known allergen protein sequence, wherein the at least one known allergen comprises Gly m 7 and the identified potential allergens comprise at least one sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15;
(b) performing sequence alignment of the at least one known allergen and potential allergens identified in step (a);
(c) selecting a consensus sequence or representative sequence based on the sequence alignment;
(d) determining a plural of candidate signature peptides based on conservative regions or domains from the sequence alignment and in silico digestion data of the consensus sequence or representative sequence selected in Step (c); and
(e) quantitating the amount of the at least one known allergen and potential allergens in the plant-based sample based on measurements of the signature peptides.
2. The method of claim 1 , wherein the quantitating step uses a column chromatography and mass spectrometry.
3. The method of claim 1 , wherein the quantitating step comprises measuring the plural of candidate signature peptides using high resolution accurate mass spectrometry (HRAM MS).
4. The method of claim 1 , wherein the quantitating step comprises calculating corresponding peak heights or peak areas of the candidate signature peptides from mass spectrometry.
5. The method of claim 1 , wherein the quantitating step comprises comparing data from high fragmentation mode and low fragmentation mode from mass spectrometry.
6. The method of claim 1 , wherein the at least one known allergen comprises Gly m 7.
7. The method of claim 1 , wherein the candidate signature peptides comprise at least one sequence selected from SEQ ID NOs: 33-43.
8. The method of claim 1 , wherein the candidate signature peptides comprise SEQ ID NO: 33, 37, or 41.
9. The method of claim 1 , wherein the plant-based sample comprises a soybean seed or part of a soybean seed.