Granted Patent B2

US 12,360,109 · App. 18/644,619 · Granted Jul 15, 2025

Inventors: Omid Farokhzad (Waban, MA); Morteza Mahmoudi (Brookline, MA); Claudia Corbo (Milan, IT)

Assignee: THE BRIGHAM AND WOMEN'S HOSPITAL, INC.

G01N33/5432C01G49/02G01N33/54326G01N33/57488G01N33/6842G01N33/6848G06F18/24G06N20/20G16B40/20B82Y35/00C01P2004/64G06F2218/20

Abstract

Claims (39)

1. A method for assaying a plasma or serum sample, comprising:

measuring proteins from the plasma or serum sample bound to magnetic particles, wherein the proteins in the plasma or serum sample are detected using mass spectrometry to measure the proteins comprising concentrations varying across a dynamic range of more than 6 orders of magnitude in the plasma or serum sample by:

(a) contacting the magnetic particles with the plasma or serum sample that comprises the proteins to allow the proteins in the plasma or serum sample to bind to the magnetic particles, wherein the magnetic particles comprise an iron oxide core, a polymer, an ammonium functional group, and a positive surface charge;

(b) separating the magnetic particles comprising the proteins from the plasma or serum sample by isolating the magnetic particles from the plasma or serum sample, thereby separating the proteins from the plasma or serum sample;

(c) washing the magnetic particles with a solution;

(d) alkylating and reducing the proteins bound to the magnetic particles;

(e) digesting the proteins from the plasma or serum bound to the magnetic particles to generate peptides; and

(f) detecting the peptides using liquid chromatography tandem mass spectrometry.

2. The method of claim 1 , wherein the washing is performed at least twice.

3. The method of claim 1 , wherein the digesting is performed using trypsin.

4. The method of claim 1 , wherein the digesting is performed using trypsin and LysC.

5. The method of claim 1 , wherein the contacting the magnetic particles with the plasma or serum sample further comprises allowing lipids from the plasma or serum to bind to the magnetic particles.

6. The method of claim 1 , further comprising purifying the peptides before detection.

7. The method of claim 6 , wherein the purifying comprises solid-phase extraction.

8. The method of claim 1 , wherein the magnetic particles are contacted with plasma.

9. The method of claim 1 , wherein the plasma or serum sample is contacted with the magnetic particles at a temperature between 10° C. and 40° C. for at least 15 minutes.

10. The method of claim 1 , wherein the magnetic particles comprise a range of sizes from about 5 μm to 8 μm in diameter.

11. The method of claim 1 , wherein the magnetic particles comprise a range of sizes from about 5 μm to 8 μm in diameter.

12. The method of claim 1 , further comprising performing (a) through (d) for a plurality of diseased plasma or serum samples and a plurality of control plasma or serum samples, and comparing the peptides detected in the diseased plasma or serum samples to the control plasma or serum samples to determine a pattern of biomarkers associated with the disease.

13. The method of claim 12 , wherein the disease is cancer.

14. The method of claim 1 , wherein the proteins in the plasma or serum sample are detected using mass spectrometry to measure the proteins comprising concentrations varying across a dynamic range of at least 10 orders of magnitude in the plasma or serum sample.

15. The method of claim 1 , wherein the polymer is cross-linked.

16. The method of claim 1 , wherein the polymer comprises a nitrogen.

17. The method of claim 1 , wherein the polymer comprises polyethylene imine.

18. The method of claim 1 , wherein the polymer comprises a secondary amine functional group.

19. The method of claim 1 , wherein the polymer comprises methacrylamide.

20. The method of claim 1 , wherein the magnetic particles are about 100 nm to 500 nm in diameter.

21. The method of claim 1 , wherein the magnetic particles comprise particles with at least one different physicochemical property.

22. The method of claim 21 , wherein the at least one different physicochemical property is size.

23. The method of claim 21 , wherein the at least one different physicochemical property is surface functionalization.

24. The method of claim 23 , wherein the magnetic particles comprise particles with carboxyl-modified silica.

25. A method for assaying a plasma or serum sample, comprising:

measuring proteins from the plasma or serum sample bound to magnetic particles, wherein the proteins in the plasma or serum sample are detected using mass spectrometry to measure the proteins comprising concentrations varying across a dynamic range of more than 6 orders of magnitude in the plasma or serum sample by:

(a) contacting the magnetic particles with the plasma or serum sample that comprises lipids and the proteins at a temperature between 10° C. and 40° C. for at least 15 minutes to allow the proteins and the lipids in the plasma or serum sample to bind to the magnetic particles, wherein the magnetic particles comprise an iron oxide core, a polymer, an ammonium functional group, and a positive surface charge;

(b) separating the magnetic particles comprising the proteins and the lipids from the plasma or serum sample by isolating the magnetic particles from the plasma or serum, thereby separating the proteins and the lipids from the plasma or serum sample;

(c) washing the magnetic particles at least twice with a solution;

(d) alkylating and reducing the proteins bound to the magnetic particles;

(e) digesting the proteins bound to the magnetic particles using trypsin to generate peptides; and

(f) detecting the peptides using liquid chromatography tandem mass spectrometry.

Continuity (8)

References Cited (399)