US 12,135,328 B2
Tandem mass tag multiplexed quantitation of post-translational modifications of proteins
Yuan Mao, Hartsdale, NY (US); and Andrew Kleinberg, Roslyn Heights, NY (US)
Assigned to Regeneron Pharmaceuticals, Inc., Tarrytown, NY (US)
Filed by Regeneron Pharmaceuticals, Inc., Tarrytown, NY (US)
Filed on Mar. 20, 2023, as Appl. No. 18/123,753.
Application 18/123,753 is a continuation of application No. 17/159,136, filed on Jan. 26, 2021, granted, now 11,639,939.
Claims priority of provisional application 62/966,151, filed on Jan. 27, 2020.
Prior Publication US 2023/0349916 A1, Nov. 2, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. G01N 33/68 (2006.01); G01N 30/72 (2006.01); G01N 30/02 (2006.01)
CPC G01N 33/6848 (2013.01) [G01N 30/7233 (2013.01); G01N 2030/027 (2013.01); G01N 2458/15 (2013.01)] 17 Claims
OG exemplary drawing
 
1. A method of quantifying multiple quality attributes of multiple samples in a single mass spectrometry (MS) run, comprising:
contacting two or more samples with a digesting solution under conditions sufficient to digest the two or more samples, wherein each sample is digested separately and the digesting solution is a Tris-free buffer solution, wherein the two or more samples comprise an antibody or antigen-binding fragment thereof;
contacting each of the two or more digested samples with a small molecule additive prior to contacting each of the two or more digested samples with a specific Tandem Mass Tag (TMT) labeling reagent, wherein the small molecule additive is selected from the group consisting of BOC—Y—OH, p-Cresol, Hydroxy-phenyl acetic acid (HPAA), hydroxy benzoic acid (HBA), acetaminophen, and p-Amino Benzoic acid (PABA);
contacting each of the two or more digested samples with the TMT labeling reagent under conditions sufficient to label peptides within each of the two or more digested samples with the specific TMT labeling reagent;
quenching labeling of peptides within each of the two or more digested samples;
combining equal volumes of the two or more labeled, digested samples into a single combined sample solution; and
analyzing the single combined sample solution by targeted mass spectral analysis, thereby allowing multiple quality attributes of the two or more samples to be quantified in a single MS run.