US 12,292,447 B2
LC-MS/MS-based methods for characterizing proteins
Susan MacIntosh, Saint Paul, MN (US); Michelle Colgrave, Brisbane (AU); Keren Byrne, St. Lucia (AU); Jo Caine, Parkville (AU); and Xue-Rong Zhou, Canberra (AU)
Assigned to Nuseed Nutritional Australia Pty Ltd, Victoria (AU); and Commonwealth Scientific and Industrial Research Organisation, Act (AU)
Appl. No. 16/492,089
Filed by Nuseed Pty Ltd., Laverton North (AU); and COMMONWEALTH SCIENTIFIC & INDUSTRIAL RESEARCH ORG., Act (AU)
PCT Filed Mar. 8, 2018, PCT No. PCT/US2018/021423
§ 371(c)(1), (2) Date Sep. 6, 2019,
PCT Pub. No. WO2018/165350, PCT Pub. Date Sep. 13, 2018.
Claims priority of provisional application 62/468,331, filed on Mar. 7, 2017.
Prior Publication US 2021/0263041 A1, Aug. 26, 2021
Int. Cl. G01N 33/68 (2006.01); G01N 30/72 (2006.01); H01J 49/00 (2006.01); G01N 30/02 (2006.01)
CPC G01N 33/6848 (2013.01) [G01N 30/7233 (2013.01); H01J 49/0036 (2013.01); G01N 2030/027 (2013.01); G01N 2333/96433 (2013.01); G01N 2333/96477 (2013.01)] 7 Claims
OG exemplary drawing
 
1. A method for characterizing the stability of a target protein in a simulated digestion assay, comprising the steps of:
(a) subjecting a target protein to pepsin digestion;
(b) obtaining a plurality of samples of peptides resulting from pepsin digestion at a plurality of time points and collecting LC-MS/MS data directly for each of the plurality of pepsin digestion samples at each of the plurality of time points without first enriching for membrane or microsomal fractions or separating the target protein by electrophoresis;
(c) subjecting the plurality of samples of pepsin digestion samples to complete trypsin digestion and collecting LC-MS/MS data directly for each of the plurality of pepsin-trypsin digestion samples after trypsin digestion is completed for each of the plurality of time points without first enriching for membrane or microsomal fractions or separating the target protein by electrophoresis; and
(d) determining from the LC-MS/MS data the kinetics of target protein digestion and the susceptibility to proteolysis of specific regions of the target protein by comparing the data from each of the plurality of time points of the pepsin digestion with the data from the corresponding dual pepsin-trypsin digestion, wherein the decline of tryptic peptides obtained from the dual pepsin-trypsin digestion is used as a proxy for intact target protein, and the appearance and disappearance of peptic peptides is used to indicate the in vitro digestibility of the target protein.