Targeted protein characterization by mass spectrometry

Patent metadata

Field	Value
Publication number	US-10877044-B2
Application number	US-201916429591-A
Country	US
Kind code	B2
Filing date	Jun 3, 2019
Priority date	Jun 6, 2018
Publication date	Dec 29, 2020
Grant date	Dec 29, 2020

Abstract

The invention provides methods for characterizing a target protein wherein a mass spectrum of digest peptides of the target protein is acquired and compared with measured reference mass spectra of digest peptides of reference proteins or of proteins of reference host cells. The comparison comprises determining similarity scores of the intensity patterns of the mass spectrum and the reference mass spectra. The characterization comprises assigning the target protein to a reference protein having a reference mass spectrum with a similarity score above a predetermined threshold.

First claim

The invention claimed is:

1. A method for characterizing a target protein, comprising the steps of:
providing a library of measured reference mass spectra of reference proteins wherein each reference mass spectrum is acquired for an enzymatic digest of one reference protein and the conditions of the enzymatic digest are substantially equal for all reference proteins;
enzymatically digesting the target protein under the same conditions used for the reference proteins;
acquiring a mass spectrum for the enzymatically digested target protein;
determining similarity scores for intensity patterns of the mass spectrum and the reference mass spectra; and
characterizing the target protein by assigning the target protein to a reference protein having a reference mass spectrum with a similarity score above a predetermined threshold.

2. The method according to claim 1 , wherein the enzymatic digest uses at least one of trypsin, Ides and Lys-C.

3. The method according to claim 2 , wherein the target protein and the reference proteins are equally denatured by a reducing agent and by a denaturing agent prior to the enzymatic digest.

4. The method according to claim 1 , wherein the mass spectrum of the digested target protein and the reference mass spectra are acquired by a MALDI-TOF mass spectrometer.

5. The method according to claim 1 , wherein the reference proteins have different amino acid sequences, the enzymatic digests of the reference proteins comprise digest peptides each having masses specific to the corresponding reference protein and the target protein is identified by assignment to one of the reference proteins.

6. The method according to claim 1 , wherein the target protein is extracted from a complex sample mixture by affinity capture.

7. The method according to claim 6 , wherein the complex mixture is one of urine, plasma, serum, spinal fluid and lysed tissue cells.

8. The method according to claim 6 , wherein an antibody is used for the affinity capture.

9. The method according to claim 1 , wherein the target protein is a biopharmaceutical.

10. The method according to claim 9 , wherein the reference proteins are different protein isoforms of the biopharmaceutical which are formed by at least one of alternative splicings, sequence variations, post-transcriptional modifications and stress-induced modifications, and the target protein is identified as one of the isoforms by assignment to one of the reference proteins.

11. The method according to claim 1 , wherein the target protein is an antibody.

12. The method according to claim 11 wherein the reference proteins are different modifications of the antibody, the target protein and the reference proteins are enzymatically digested into domains and the target protein is identified to be modified by assignment to one of the reference proteins.

13. The method according to claim 12 , wherein the modifications are at least one of glycosylation, oxidation, acetylation and amidation.

14. The method according to claim 13 , wherein the enzyme IdeS is used to prepare Fc/2 antibody domains for glycoprofiling.

15. The method according to claim 12 , wherein one reference protein is the antibody and the target protein is identified to be the antibody by assignment to this reference protein.

16. The method according to claim 1 , wherein redundant mass signals which are present in a majority of the measured reference mass spectra are removed, and reduced reference mass spectra are provided and/or used for the determining step.

17. The method according to claim 16 , wherein the reference proteins have different amino acid sequences, the enzymatic digest of the reference proteins comprises digest peptides with masses which are characteristic for the corresponding reference protein and reduced reference mass spectra comprise only the characteristic mass signals.

18. The method according to claim 16 , wherein the reduced reference mass spectra additionally comprise some abundant mass signals which are present in substantially all reduced reference mass spectra and the number of the abundant mass signals is lower than the number of the characteristic mass signals.

19. The method according to claim 1 , wherein the similarity scores are determined by a cosine similarity measure or a cross-correlation.

20. A method for characterizing proteins of target host cells, comprising the steps of:
providing a library of measured reference mass spectra of proteins of reference host cells wherein each reference mass spectrum comprises mass signals of digest peptides which are generated by an enzymatic digest of multiple proteins of one type of reference host cells and are extracted by affinity capture using multiple affinity agents after the enzymatic digest;
enzymatically digesting the proteins of the target host cells under the same conditions used for the proteins of the reference host cells and then extracting the digest peptides by affinity capture using the multiple affinity agents;
acquiring a mass spectrum for the extracted digest peptides of the target host cells;
determining similarity scores of the intensity pattern of the mass spectrum and the reference mass spectra; and
characterizing the target host cells by assigning the proteins of the target host cells to a type of reference host cell having a reference spectrum with a similarity score above a predetermined threshold.

Assignees

• Bruker Daltonik Gmbh

Inventors

• Suckau Detlev
• Resemann Anja
• Evers Waltraud

Classifications

• H01J49/26

  Mass spectrometers or separator tubes · CPC title

• G01N33/6848 Primary

  Methods of protein analysis involving mass spectrometry · CPC title

• G01N30/72

  Mass spectrometers {(mass spectrometers per se H01J49/00)} · CPC title

• G01N27/623 Primary

  combined with mass spectrometry · CPC title

• H01J49/40

  Time-of-flight spectrometers (H01J49/36 takes precedence) · CPC title