US 12,292,448 B2
Quantitative analysis of proteins
Kevin B. Ray, Ballwin, MO (US); Pegah Jalili, St. Louis, MO (US); Jeffrey L. Turner, Maryville, IL (US); Nicolas Caffarelli, St. Louis, MO (US); and Yue Lu, St. Louis, MO (US)
Assigned to Sigma-Aldrich Co. LLC, St. Louis, MO (US)
Appl. No. 16/971,286
Filed by Sigma-Aldrich Co. LLC, St. Louis, MO (US)
PCT Filed May 8, 2019, PCT No. PCT/US2019/031271
§ 371(c)(1), (2) Date Aug. 19, 2020,
PCT Pub. No. WO2019/226347, PCT Pub. Date Nov. 28, 2019.
Claims priority of provisional application 62/675,989, filed on May 24, 2018.
Prior Publication US 2020/0378986 A1, Dec. 3, 2020
Int. Cl. G01N 33/543 (2006.01); G01N 30/02 (2006.01); G01N 33/538 (2006.01); G01N 33/549 (2006.01); G01N 33/563 (2006.01); G01N 33/68 (2006.01)
CPC G01N 33/6848 (2013.01) [G01N 30/02 (2013.01); G01N 33/538 (2013.01); G01N 33/549 (2013.01); G01N 33/563 (2013.01); G01N 2030/027 (2013.01); G01N 2333/976 (2013.01)] 25 Claims
 
1. A method for detecting and/or quantitating a target protein in a biological sample, the method comprising:
a) adding the biological sample comprising the target protein to an immobilized capture entity to capture the target protein, thereby forming an immobilized target protein;
b) adding a denaturation solution comprising an organic solvent to the immobilized target protein to form a denatured immobilized target protein;
c) digesting the denatured immobilized target protein at a temperature from 40° C. to 80° C. with a digestion solution comprising a protease to form a solution comprising proteolytic peptides, wherein said method is devoid of a reducing step and an alkylating step prior to step (c); and
d) analyzing the solution comprising proteolytic peptides by a mass spectrometry-based technique to detect and/or quantitate the target protein in the biological sample, wherein the analyzing utilizes at least one internal standard protein or peptide that is added during one step of the method, and wherein said solution comprising proteolytic peptides is not subject to a clean-up process prior to analyzing the solution by the mass spectrometry-based technique.