title: Comparison of Gluten Extraction Protocols Assessed by LC-MS/MS Analysis.
abstract: The efficiency of gluten extraction is of critical importance to the results derived from any analytical method for gluten detection and quantitation, whether it employs reagent-based technology (antibodies) or analytical instrumentation (mass spectrometry). If the target proteins are not efficiently extracted, the end result will be an under-estimation in the gluten content posing a health risk to people affected by conditions such as celiac disease (CD) and nonceliac gluten sensitivity (NCGS). Five different extraction protocols were investigated using LC-MRM-MS for their ability to efficiently and reproducibly extract gluten. The rapid and simple "IPA/DTT" protocol and related "two-step" protocol were enriched for gluten proteins, 55/86% (trypsin/chymotrypsin) and 41/68% of all protein identifications, respectively, with both methods showing high reproducibility (CV < 15%). When using multistep protocols, it was critical to examine all fractions, as coextraction of proteins occurred across fractions, with significant levels of proteins existing in unexpected fractions and not all proteins within a particular gluten class behaving the same.
authors.affiliation: CSIRO Agriculture and Food , 306 Carmody Road, St. Lucia, Queensland 4067, Australia.
authors.forename: Azadeh
authors.initials: A
authors.lastname: Fallahbaghery
authors.affiliation: CSIRO Agriculture and Food , 306 Carmody Road, St. Lucia, Queensland 4067, Australia.
authors.forename: Wei
authors.initials: W
authors.lastname: Zou
authors.affiliation: CSIRO Agriculture and Food , 306 Carmody Road, St. Lucia, Queensland 4067, Australia.
authors.forename: Keren
authors.initials: K
authors.lastname: Byrne
authors.affiliation: CSIRO Agriculture and Food , GPO Box 1700, Canberra, ACT 2601, Australia.
authors.forename: Crispin A
authors.initials: CA
authors.lastname: Howitt
authors.affiliation: CSIRO Agriculture and Food , 306 Carmody Road, St. Lucia, Queensland 4067, Australia.
authors.forename: Michelle L
authors.identifier: http://orcid.org/0000-0001-8463-805X
authors.initials: ML
authors.lastname: Colgrave
mesh_terms: D005591:Chemical Fractionation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005983:Glutens / Q000032:analysis* / Q000302:isolation & purification*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods; D014908:Triticum / Q000737:chemistry*
chemical_list: D005983:Glutens
keywords: cereal; contamination; gluten; mass spectrometry; multiple reaction monitoring (MRM); quantitation