Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Summary

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Abstract

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Introduction

Despite the completion of the human genome sequencing, significant progress has not been made in identifying biomarkers predictive of early stage disease, or that correlate with therapeutic outcome, or prognosis¹. One reason for this lack of progress is that many potentially significant biomarkers exist at a concentration below the detection limit of conventional mass spectrometry and other biomarker discovery platforms. Mass spectrometry (MS) and Multiple Reaction Monitoring (MRM) have a detection sensitivity typically greater than 50 ng/ml while the majority of the analytes measured by immunoassays in a clinical laboratory fall in the range between 50 pg/ml and 10 ng/ml. This means that many biomarkers, particularly in the early stage of a disease cannot be detected by conventional MS and MRM². In addition the presence of high-abundance proteins such as albumin and immunoglobulin in complex biological fluids often mask by billion-fold excess low-abundance, low molecular weight proteins and peptides^{3, 4}. For this reason several sample preparatory steps are required prior to mass spectrometry sequencing and identification. One such preparatory step employs the depletion of high-abundance proteins with commercially available depletion columns^5-8. Unfortunately this step leads to the reduction of the yield of candidate biomarkers because they are often non-covalently associated with carrier proteins that are being removed. Another challenge is represented by the stability of candidate biomarkers ex-vivo once the samples are collected. Proteins are subject to degradation by endogenous or exogenous proteases⁹. Hydrogel nanoparticles can transcend these critical challenges by amplifying the putative biomarker concentration to a level within the range of the assay, while protecting the protein from degradation^10-13.

It’s important to note that LMW proteins in blood are a mixture of small intact proteins as well as fragments of large proteins. Tissue-derived proteins larger than 60 kDa are too large to passively enter the blood stream through the vascular basement membrane, but they can be represented in blood as peptides or protein fragments¹⁴. Our goal is to measure novel circulating biomarkers that can be candidates for early detection of disease, patient stratification for therapy, and monitoring the response to therapy. Our nanoparticles are created to selectively exclude high abundance immunoglobulins and albumin, while simultaneously capturing smaller proteins and peptides and concentrating them up to 100-fold depending on the starting volume.

Our group identified a set of small organic dyes which can successfully act as high affinity molecular baits for proteins and peptides. Protein-dye binding is thought to be due to a combination of hydrophobic and electrostatic interactions. The aromatic rings on the dye interleave with proteins via hydrophobic pockets on the protein surface¹¹.

The baits, depending on their chemistry, show a particular affinity for selected classes of analytes. The baits compete with the carrier proteins, such as albumin, for the proteins or peptides. The low-molecular weight proteins/peptides become trapped in the particle. High-molecular weight proteins such as albumin and immunoglobulin are prevented from entering the particle because of the sieving capability due to the restrictive pore of the hydrogel¹¹(Figure 1).

Hydrogel nanoparticles are synthesized by precipitation polymerization initiated by ammonium persulfate¹¹. N-isopropylacrylamide (NIPAm), co-monomers of acrylic acid (AAc) and allylamine (AA) and cross-linker N,N’-Methylenebisacrylamide (BIS) are allowed to react at 70 °C for 6 hr in dilute conditions^{11, 13}. The high protein binding affinity of poly(N-isopropylacrylamide-co-acrylic acid) (poly(NIPAm-co-AAc) nanoparticlesis achieved by covalently incorporating amino-containing dyes (i.e., sulfonatedanthraquinonetriazine dyes) into the nanoparticles through an amidation reaction performed in aqueous or organic solvents depending on the hydrophilic/hydrophobic characteristics of the dyes^{11, 13}. Nucleophilic substitution of the amine groups in the nanoparticle with the chloride atom of an anthraquinonetriazine dye is utilized to create dye-containing poly(NIPAm-co-Allylamine) (AA) nanoparticles^{11, 12}. A two-step polymerization process is utilized to create hydrogel nanoparticles containing an outer shell of vinylsulfonic acid (VSA)^{11, 13}.

Hydrogel nanoparticles can be applied to various biological fluids, including whole blood, plasma, serum, cerebrospinal fluid, sweat, and urine. In one step, in solution, the nanoparticles perform a rapid (within minutes) sequestration and concentration of low molecular weight analytes^{10, 11, 13, 15-18}. Proteins are subsequently eluted from the nanoparticles and detected using western blotting^19-21, mass spectrometry^{10, 11, 13, 15, 18, 22, 23}, immunoassays/ELISA^{10, 11, 15, 18}, or reverse phase protein microarray^{16, 24} assays. Nanoparticles functionalized with chemical bait, and presenting a core or core shell architecture, capture and concentrate proteins based on the bait/shell physicochemical properties. Different dyes incorporated into the nanoparticles will therefore capture different subsets of proteins with varying efficiency based on the dye affinity, pH of the solution, and the presence/absence of competing high-abundant proteins¹³. Furthermore, the quantity of nanoparticles in relation to the volume of the solution will affect the protein yield from the nanoparticles. These aspects of hydrogel nanoparticle harvesting are demonstrated using three different nanoparticle baits for harvesting proteins from plasma samples which contain high amounts of protein, and from urine samples which typically do not contain large amounts of protein. In this protocol we demonstrate harvesting and concentrating tumor necrosis factor alpha (TNFα) from plasma samples using poly(NIPAm-co-AAc), Poly(NIPAm/dye), and core- shell nanoparticles (Poly(NIPAm-co-VSA)). Poly(NIPAm/dye) nanoparticles are shown to concentrate Mycobacterium species antigen that was added to human urine samples, to mimic Mycobacterium tuberculosis infected individuals.

Protocol

Human plasma and urine was collected from healthy volunteer donors, with written informed consent, following George Mason University Institutional Review Board approved protocols. Donors were equally distributed between Caucasian males and females between the ages of 25 and 42. Samples were analyzed individually and were not pooled.

1. Nanoparticle Processing of Serum or Plasma Samples

Potential low abundant biomarkers in plasma are captured, in solution, with hydrogel nanoparticles. The particles are added to the plasma, incubated, separated by centrifugation, washed, and the captured proteins are eluted. The eluted proteins are dried under nitrogen flow for downstream mass spectrometry sequencing and identification.

1. Dilute 500 µl of serum 1:2 with 50 mM TrisHCl pH 7 (500 µl serum + 500 µl TrisHCl) in a microcentrifuge tube.
2. Add 500 µL of poly(NIPAm/AAc) core nanoparticles. Incubate for 15 min at RT.
3. Spin the sample at 16,100 x g, 25 °C for 10 min in a centrifuge equipped with a fixed-angle rotor. Remove and discard the supernatant.
4. Add 500 µl sodium thiocyanate (25 mM) to the pellet. Resuspend the nanoparticles by vigorously pipetting up and down multiple times.
5. Spin the sample at 16,100 x g, 25 °C for 10 min in a centrifuge with a fixed angle rotor. Remove and discard the supernatant.
6. Add 500 µl of MilliQ water to the nanoparticle pellet. Resuspend the nanoparticles by vigorously pipetting up and down multiple times.
7. Spin the sample at 16,100 x g, 25 °C for 10 min in a centrifuge with a fixed angle rotor. Remove and discard the supernatant.
8. Prepare fresh elution buffer: Add 700 µl acetonitrile to 300 µl ammonium hydroxide in a clean tube. Caution: Ammonium hydroxide is corrosive. Use with appropriate ventilation. Note: The elution buffer must be prepared immediately before use. Do not store the elution buffer.
9. Add 300 µl of elution buffer to the nanoparticle pellet. Resuspend the nanoparticles by vigorously pipetting up and down multiple times. Incubate for 15 min at RT.
10. Spin the sample at 16,100 x g, 25 °C for 10 min in a centrifuge with a fixed angle rotor. Remove and save the eluate in a clean, labeled microcentrifuge tube.
11. Repeat steps 1.9 – 1.10. Combine the two eluates into one microcentrifuge tube.
12. Dry the eluates under nitrogen flow in a nitrogen evaporator manifold at 42.1 °C, with air flow set to 8 (Figure 2F).
13. Store the dried eluate at RT for O/N storage, or at -20 °C for long term storage, prior to mass spectrometry, western blotting, or ELISA assays (optional).

2. Nanoparticle Processing of Urine Samples

Normal urine contains less than 30 mg/dl protein and less than 1+ blood. However, many diseases/conditions may alter the normal levels of urine protein and blood. To aid in determining the optimal volume of nanoparticles to add to the urine sample, a urinalysis is performed prior to nanoparticle harvesting. Urine biomarkers may exist in extremely low concentrations, which may require optimizing the ratio of nanoparticles to urine volume. This procedure describes nanoparticle harvesting of urine samples for downstream western blot analysis.

1. Collect urine samples in a clean, dry plastic cup. A 22 ml minimum volume is required. Store urine specimens at -80 °C until ready to analyze.
2. Thaw the frozen urine at RT or at 4 °C O/N. Mix briefly on a vortex mixer. Pour at least 22 ml of urine into a 50 ml conical bottom polypropylene tube.
3. Spin the urine in a centrifuge with a swing-out rotor at 3,700 x g for 15 min. Decant the urine, without disturbing the pellet, into a clean 50 ml conical bottom polypropylene tube. Discard the pellet.
4. Perform urinalysis using a multi-analyte “urine dipstick” reagent strip. Note: store the reagent strips in a tightly closed container away from direct sunlight and humidity^{17, 18}.
   1. Lay the reagent strip, face-up, on a clean, dry paper towel.
   2. Use a disposable pipette to aspirate 1 ml of the urine prepared in step 2.3.
   3. Set a timer for 2 min but do not start it. Quickly dispense 1 - 2 drops of urine on each test pad of the reagent strip. Immediately start the timer. Note: Do not submerge the reagent strip directly in the urine container. The dye/chemical indicators in the reagent strip can potentially leach out of the reagent strip into the urine container.
   4. At the time indicated on the reagent strip container, record the qualitative results for the various analytes on the reagent strip by comparing the color of the individual reagent strips to the corresponding color-coded indicators on the reagent container. Typical normal urine results are: (normal or negative) for blood, protein, leukocytes, nitrite, glucose, ketone, bilirubin, and urobilinogen; Urine pH (5.5 - 7.0); specific gravity (1.001 - 1.020). Note: High protein levels in a urine specimen may compete with the protein of interest for binding sites on the nanoparticles. To maximize protein harvesting with nanoparticles, the nanoparticle volume/urine volume ratio can be adjusted to either limit competition from high abundance proteins, or can be optimized to harvest proteins that exist in very low concentrations. If the urine protein value is 1+ or greater, add 2x volumes of nanoparticles in step 2.5 below. If the analyte of interest is a very low abundant protein, it may be necessary to increase the volume of nanoparticles up to 2 ml (Figure 3).
5. Transfer 20 ml of the clarified urine from step 2.3 into a clean 50 ml conical bottom polypropylene tube. Do not disturb any debris/pellet that may be in the bottom of the urine tube. Add 200 µl of nanoparticles to the 20 ml urine sample. Mix briefly on a vortex mixer. Note: If the urine protein value is 1+ or greater, add 400 µl of nanoparticles to 20 ml urine.
6. Incubate the urine/nanoparticle mixture for 30 min at RT, without rocking/mixing.
7. Spin the urine/nanoparticle suspension in a centrifuge equipped with a swing-out rotor at 3,700 x g for 10 min. Note: If a pellet is not visible following centrifugation, spin the samples for an additional 2 – 7 min.
8. Remove and discard the supernatant. Add 500 µl MilliQ water to the nanoparticle pellet.
9. Resuspend the nanoparticles by vigorously pipetting up and down multiple times. Transfer the nanoparticle solution to a clean 1.5 ml microcentrifuge tube.
10. Spin the nanoparticles in a centrifuge equipped with a fixed-angle rotor at 16,100 x g for 10 min. Note: If a pellet is not visible following centrifugation, spin the samples for an additional 2 – 7 min.
11. Remove and discard the supernatant.
12. Repeat steps 2.8 and 2.11 (twice) with 200 µl 18 MilliQ water to wash the nanoparticles.
13. Prepare fresh elution buffer: Add 970 µl acetonitrile to 30 µl ammonium hydroxide in a clean tube. Caution: Ammonium hydroxide is corrosive. Use with appropriate ventilation. Note: The elution buffer must be prepared immediately before use. Do not store the elution buffer.
14. Add 20 μl elution buffer to the nanoparticle pellet. Resuspend the nanoparticles by vigorously pipetting up and down multiple times. Incubate for 15 min at RT.
15. Spin the nanoparticle samples at 16,100 x g for 10 min. Remove and save the supernatant in a clean, 1.5 ml microcentrifuge tube. Do not disrupt the nanoparticle pellet. Discard the pellet in the biohazard trash.
16. Place the samples in a rack in a chemical fume hood. Open the caps and incubate at RT for 30 min. Alternatively, place the samples under nitrogen flow at 40 °C until dry (1 - 2 hr).
17. Add 15 µl Tris-glycine SDS sample buffer (2x) to the samples. Heat at 100 °C in a dry heat block for 5 min with the caps open or until the volume left in the tube is no more than 20 µl. Note: do not use a boiling water bath. The humidity from the water bath will prevent evaporation of the buffer.
18. Place the cap on the tubes. Remove the tubes from the heat block. The sample can be stored at -80 °C or used immediately for downstream western blot analysis.

Results

Hydrogel Nanoparticle Size and Uniformity

Poly(NIPAm-AAc) particles have been produced with extremely high yield and reproducibility between and within batches. The particles have very good colloidal stability at RT during the time required for capture, storage, and elution of proteins (at least 48 hr), and nanoparticle precipitation has not been observed (Figure 1)¹¹. The colloidal stability may be very important for rapid protein/peptide uptake by...

Discussion

Clinical Relevance

A serum or plasma sample is thought to contain low-abundance circulating proteins and peptides which can provide a rich source of information regarding the state of the organism as a whole. Despite the promise of serum proteomics, there are three fundamental and serious physiologic barriers thwarting biomarker discovery and translation to clinical benefit^{10, 11, 16, 25}.

1. Important diagnostic biomarkers may exist in ex...

Materials

Name	Company	Catalog Number	Comments
hydrogel nanoparticles	Ceres Nanoscience	CS003	NanoTrap ESP particles
18 MΩ-cm water			Type 1 reagent grade water
Tris HCl, 50mM pH7.0	VWR	IC816116	50mM, pH 7
Acetonitrile	BDH	BDH1103-4LP	available from VWR
Ammonium Hydroxide NH4OH	BDH	BDH3014	available from VWR, assayed at 28-30% NH3
sodium thiocyanate 25mM	Acros Organics	419675000	for serum/plasma samples
Multi-analyte Urine Reagent Strips	Siemens	2161	for urine samples
Tris-Glycine SDS Sample Buffer (2X)	Life Technologies	LC2676	use at room temperature to prevent SDS from precipitating
Dry bath incubator (100 oC) with heating block	Barnstead	11-715-125DQ	do not substitute a boiling water bath
Nitrogen evaporator manifold	Organomation Associates	Microvap118	for serum/plasma samples
Centrifuge, swing-out rotor	Sorvall	Legend series	50ml tube capacity, rcf 3700 x g
Centrifuge, fixed angle rotor	Eppendorf	5424	1.7ml microcentrifuge capacity, rcf 16,000 x g
50ml conical centrifuge tubes	Fisher Scientific	14-432-22	with screw caps for urine samples
1.5ml microcentrifuge tubes	Eppendorf	22363204
Disposable plastic transfer pipettes	Fisher Scientific	13-711-7M	at least 1ml capacity
Vortex mixer	Fisher Scientific	50-949-755
Timer	Fisher Scientific	S04782	seconds/minutes