Granted Patent B2

US 12,287,331 · App. 18/460,474 · Granted Apr 29, 2025

Inventors: Hongwei Xia (Fremont, CA); Lyndal Hesterberg (Loveland, CO); Michael Figa (San Mateo, CA); Xiaoyan Zhao (Foster City, CA); Gregory Troiano (Pembroke, MA); William Manning (Redwood City, CA); John Blume (Bellingham, WA); Omid Farokhzad (Waban, MA); Matthew McLean (Redwood City, CA); Craig Stolarczyk (San Mateo, CA); Marwin Ko (Redwood City, CA); Theodore Platt (Danville, CA)

Assignee: Seer, Inc.

G01N33/54326G01N33/54346G01N33/551G01N33/6848

Abstract

Claims (41)

1. A high-throughput method of preparing to identify proteins in a biological sample, the method comprising:

(a) incubating magnetic microparticles with the biological sample to form protein coronas comprising proteins, the magnetic microparticles comprising: (i) silica, (ii) carboxylate functional groups, (iii) negative surface charges from −30 mV to −10 mV in zeta potential, wherein the zeta potential is measurable with a concentration of 0.02 wt % in a solution in 5% pH 7.4 PBS at about 25° C. with an about 1 minute equilibration time on a Zetasizer Nano ZS (Malvern Instruments) in the absence of biological sample, and (iv) a heterogeneous distribution of sizes including from about 1 μm to about 10 μm in diameter, wherein the magnetic microparticles are configured to form protein coronas having at least 200 unique protein groups that can be identified using mass spectrometry;

(b) magnetically isolating the magnetic microparticles, by applying an external magnetic field, from unbound protein in the biological sample to enrich the proteins in the protein coronas;

(c) washing the enriched proteins and the magnetic microparticles after magnetically isolating the magnetic microparticles;

(d) incubating the enriched proteins in the protein coronas by adding a denaturing reagent to the protein coronas and the magnetic microparticles;

(e) digesting the enriched proteins in the protein coronas to generate digested peptides by adding a digestion reagent to the protein coronas and the magnetic microparticles; and

(f) purifying the digested peptides.

2. The method of claim 1 , wherein said purifying the digested peptides comprises binding the digested peptides to a solid phase, washing the bound digested peptides, and eluting the washed and bound digested peptides.

3. The method of claim 1 , further comprising using a stop reagent to stop the digestion reagent.

4. The method of claim 1 , wherein the digestion reagent comprises trypsin.

5. The method of claim 4 , wherein the digestion reagent further comprises LysC.

6. The method of claim 1 , wherein the magnetic microparticles are configured to form protein coronas having at least 500 unique protein groups that can be identified using mass spectrometry.

7. The method of claim 1 , wherein the magnetic microparticles comprise a polydispersity index greater than 0.5.

8. The method of claim 1 , further comprising assaying, using mass spectrometry, the purified digested peptides to identify the unique protein groups from the enriched proteins.

9. The method of claim 8 , wherein the assaying yields a percent quantile normalized coefficient (QNCV) of variation of 20% or less, as determined by comparing a peptide mass spectrometry feature from at least three full-assay replicates.

10. The method of claim 1 , wherein said washing the enriched proteins and the magnetic microparticles is performed at least two times.

11. The method of claim 1 , wherein the biological sample comprises plasma or serum.

12. The method of claim 1 , wherein the magnetic microparticles are incubated with the biological sample at a basic pH.

13. The method of claim 1 , wherein (a)-(f) is performed in about 4-6 hours.

14. A high-throughput method of preparing to identify proteins in a biological sample, the method comprising:

(a) incubating magnetic particles with the biological sample to form protein coronas comprising proteins, the magnetic particles comprising at least two distinct particle types, wherein the first particle type comprises silica and having negative surface charges from −30 mV to −10 mV in zeta potential, and the second particle comprises positive surface charges, wherein the zeta potential of the magnetic microparticles is measurable with a concentration of 0.02 wt % in a solution in 5% pH 7.4 PBS at about 25° C. with an about 1 minute equilibration time on a Zetasizer Nano ZS (Malvern Instruments) in the absence of biological sample and wherein the magnetic particles are configured to form protein coronas having at least 400 unique protein groups that can be identified using mass spectrometry;

(b) magnetically isolating the magnetic particles, by applying an external magnetic field, from unbound protein in the biological sample to enrich the proteins in the protein coronas;

(c) washing the enriched proteins and the magnetic particles at least two times after the magnetically isolating of the magnetic particles;

(d) incubating the enriched proteins in the protein coronas by adding a denaturing reagent to the protein coronas and the magnetic particles;

(e) digesting the enriched proteins in the protein coronas to generate digested peptides by adding a digestion reagent to the protein coronas and the magnetic particles; and

(f) purifying the digested peptides,

wherein said purifying the digested peptides comprises binding the digested peptides to a solid phase, washing the bound digested peptides, and eluting the washed and bound digested peptides, and wherein the biological sample is plasma or serum.

15. The method of claim 14 , further comprising using a stop reagent to stop the digestion reagent.

16. The method of claim 14 , wherein the digestion reagent comprises trypsin.

17. The method of claim 16 , wherein the digestion reagent further comprises LysC.

18. The method of claim 14 , wherein the magnetic particles are configured to form protein coronas having at least 1000 unique protein groups that can be identified using mass spectrometry.

19. The method of claim 14 , wherein the magnetic particles comprise a polydispersity index less than 0.5.

20. The method of claim 14 , further comprising assaying, using mass spectrometry, the purified digested peptides to identify the unique protein groups from the enriched proteins.

21. The method of claim 14 , wherein the magnetic particles further comprise carboxylate functional groups.

22. The method of claim 14 , wherein the magnetic particles are incubated with the biological sample at a basic pH.

23. The method of claim 14 , wherein (a)-(f) is performed in 4-6 hours.

24. The method of claim 14 , wherein the second distinct particle type comprises a poly (N-(3-(dimethylamino) propyl) methacrylamide) (PDMAPMA) coating.

25. The method of claim 14 , wherein the magnetic particles are incubated with the biological sample and a Tris buffer.

26. The method of claim 14 , wherein the first distinct particle type is a nanoparticle and the second distinct particle type is a microparticle.

27. The method of claim 26 , wherein the first distinct particle type comprises an iron oxide core.

28. The method of claim 27 , wherein the second distinct particle type comprises a non-magnetic core.

Assignments (1)

Continuity (6)

References Cited (400)