US-12618851-B2 - Systems and methods for sample preparation, data generation, and protein corona analysis

Abstract

Systems and methods for automated sample preparation and processing of protein corona are described herein, as well as its application in the discovery of advanced diagnostic tools as well as therapeutic agents.

Inventors

• William Manning
• Young Kim
• Brandon Kwan-Leong
• Hope Liou
• Xiaoyan Zhao
• Daniel Hornburg
• Martin Goldberg

Assignees

• SEER, INC.

Dates

Publication Date

20260505

Application Date

20240108

Claims (20)

1. 1 . An automated system for sample preparation, the system comprising: (a) a fluid transfer unit comprising a multichannel fluid transfer instrument for transferring fluids between units within the system; (b) a multi-well plate, operably coupled to the fluid transfer unit; (c) a composition within at least one well of the multi-well plate comprising (i) at least 10 microliters of plasma or serum, (ii) an aqueous buffer, and (iii) a plurality of magnetic microspheres, wherein the plurality of magnetic microspheres comprise iron oxide and further comprise a size of least 1 micron, and wherein the plurality of magnetic microspheres are configured to bind to at least 300 different proteins from the plasma or serum; (d) a magnet configured to immobilize one or more of the plurality of magnetic microspheres, wherein the magnet is removably coupled to the multi-well plate; and (e) a control unit comprising one or more processors programmed to perform steps comprising: i. incubating the multi-well plate such that proteins from the plasma or serum bind the plurality of magnetic microspheres, wherein the incubating is performed for at least 20 minutes; ii. removing a supernatant from the at least one well of the multi-well plate to isolate proteins bound to the plurality of magnetic microspheres; iii. washing the proteins bound to at least one of the plurality of magnetic microspheres at least twice to obtain washed proteins bound to the at least one of the plurality of magnetic microspheres; and iv. preparing a population of peptides for mass spectrometry by desorbing and digesting the washed proteins bound to the at least one of the plurality of magnetic microspheres.
2. 2 . The automated system of claim 1 , wherein the plasma or serum is diluted with a diluent no more than 8-fold with the aqueous buffer.
3. 3 . The automated system of claim 1 , wherein the plasma or serum comprises plasma.
4. 4 . The automated system of claim 1 , wherein the plurality of magnetic microspheres comprises microspheres having a size of at least 1000 nm.
5. 5 . The automated system of claim 1 , wherein the plurality of magnetic microspheres comprises a negative surface charge.
6. 6 . The automated system of claim 5 , wherein the plurality of magnetic microspheres comprises a carboxyl functionalization.
7. 7 . The automated system of claim 1 , wherein the plurality of magnetic microspheres comprises a positive surface charge.
8. 8 . The automated system of claim 7 , wherein the plurality of magnetic microspheres comprises an ammonium functionalization.
9. 9 . The automated system of claim 8 , wherein the ammonium functionalization is a quaternary ammonium functionalization.
10. 10 . The automated system of claim 8 , wherein the plurality of magnetic microspheres comprise polyethylene imine.
11. 11 . The automated system of claim 1 , wherein the digesting comprises contacting the washed proteins bound to the at least one of the plurality of magnetic microspheres with a solution comprising trypsin.
12. 12 . The automated system of claim 1 , wherein the plurality of magnetic microspheres are configured to bind to at least 500 different proteins from the plasma or serum.
13. 13 . The automated system of claim 1 , wherein the plurality of magnetic microspheres comprises a homogeneous size distribution.
14. 14 . The automated system of claim 1 , wherein the plurality of magnetic microspheres comprises a heterogeneous size distribution.
15. 15 . The automated system of claim 1 , wherein the at least one well of the multi-well plate comprises 1 mg/mL to 3 mg/mL of the plurality of magnetic microspheres.
16. 16 . The automated system of claim 1 , wherein the plurality of magnetic microspheres comprises a non-polymeric outer shell.
17. 17 . The automated system of claim 1 , wherein the preparing in step (iv) further comprises incubating the washed proteins and the plurality of magnetic microspheres with chemical reagents at 95° C. for 10 minutes to lyse, reduce, and alkylate the washed proteins.
18. 18 . A method comprising obtaining peptides from the plasma or serum using the automated instrument of claim 1 .
19. 19 . The method of claim 18 , further comprising performing mass spectrometry on the peptides to identify at least 300 proteins in the plasma or serum.
20. 20 . The method of claim 19 , further comprising purifying the peptides using solid phase extraction.

Description

CROSS-REFERENCE The present application is a continuation of U.S. Non-Provisional application Ser. No. 18/365,674, filed Aug. 4, 2023, which is a continuation of U.S. Non-Provisional application Ser. No. 18/178,288, filed Mar. 3, 2023, which is a continuation of U.S. Non-Provisional application Ser. No. 17/216,523, filed Mar. 29, 2021, issued as U.S. Pat. No. 11,630,112, on Apr. 18, 2023, which is a continuation of International Application No. PCT/US2020/044908, filed Aug. 4, 2020, which claims the benefit of U.S. Provisional Patent Application No. 62/883,107, filed Aug. 5, 2019, each of which is herein incorporated by reference in its entirely. BACKGROUND Broad scale implementation of proteomic information in science and medicine has lagged behind genomics in large part because of complexities inherent in protein molecules themselves, necessitating complex workflows that limit the scalability of such analyses. Disclosed herein are systems, methods and kits for rapid and automated sample preparation, processing of proteomic data and the identification of key biomarkers associated with diseased states. SUMMARY The present disclosure provides automated systems, methods and kits for protein corona preparation and analysis. In some aspects, the present disclosure provides an automated apparatus for generating a subset of biomolecules from a complex biological sample, the automated apparatus comprising: (i) a substrate comprising a plurality of partitions, wherein the plurality of partitions comprises a plurality of particles; (ii) a sample storage unit comprising the complex biological sample; and (iii) a loading unit that is movable at least across the substrate, wherein the loading unit transfers one or more volumes of the complex biological sample in the sample storage unit to the plurality of partitions on the substrate, thereby contacting the plurality of particles in the plurality of partitions with biomolecules of the complex biological sample to form biomolecule coronas, thereby generating the subset of biomolecules of the complex biological sample, and wherein a dynamic range of the subset of biomolecules is compressed relative to a dynamic range of biomolecules present in the complex biological sample. In some embodiments, the substrate is a multi-well plate. In some embodiments, the subset of biomolecules comprises at least 20% to at least 60% of the types of biomolecules from the complex biological sample within a 6 order of magnitude concentration range. In some embodiments, the subset of biomolecules comprises at least 20% to at least 60% of the types of proteins from the complex biological sample within a 6 order of magnitude concentration range. In some embodiments, the automated apparatus generates the subset of biomolecules from a complex biological sample in less than 7 hours. In some embodiments, the automated apparatus comprises an incubation element that agitates or heats volumes of the plurality of particles within volumes of the complex biological sample in the plurality of partitions. In some embodiments, the incubation element is configured to shake, mix, stir, spin, vibrate, be static, or any combination thereof. In some embodiments, the wherein the incubation element is configured to heat and/or incubate the substrate to a temperature between about 20° C. and about 100° C. In some embodiments, the plurality of partitions is at least partially covered or sealed. In some embodiments, a partition from among the plurality of partitions is covered or sealed. In some embodiments, the automated apparatus comprises the ability to add or remove a lid on the substrate, wherein the lid covers at least one of the partitions from among the plurality of partitions. In some embodiments, the automated apparatus comprises a unit comprising a resuspension solution. In some embodiments, the resuspension solution comprises Tris EDTA 150 mM KCl 0.05% CHAPS buffer. In some embodiments, the resuspension solution comprises 10 mM Tris HCl pH 7.4, 1 mM EDTA. In some embodiments, the apparatus comprises a unit comprising a denaturing solution. In some embodiments, the denaturing solution comprises a protease. In some embodiments, the denaturing solution comprises a reductant, a methylating agent, guanidine, urea, sodium deoxycholate, acetonitrile, or any combination thereof. In some embodiments, the denaturing solution generates an average peptide fragment with a mass of less than 4600 Daltons. In some embodiments, the loading unit comprises a plurality of pipettes. In some embodiments, the loading unit is configured to dispense 10 uL to 400 uL of a solution into one or more partitions of the plurality of partitions. In some embodiments, the loading unit is configured to dispense 5 uL to 150 uL of a solution into one or more partitions of the plurality of partitions. In some embodiments, the loading unit is configured to dispense 35 uL to 80 uL of a solution into one or more partitions of the plurality of par