Journal of the American Chemical Society Table 1. Washing and Elution Buﬀers for Each Kind of Nanoparticle chemical bait in the particles Cibacron Blue F3GA, Remazol Brilliant Blue R Disperse Yellow 9, Disperse Yellow 3, Disperse Orange 3, Pararosaniline base, Pigment Red 177, acid black 48, Acid blue 22, Acid black 1; Brilliant Blue R250, Toluidine Blue, Rhodamine 123, Disperse Blue 3, VSA, AAc washing buﬀer 0.25 M NaSCN Twice 0.5 PBS and 0.5 PBS/20% acetonitrile elution buﬀer 70% acetonitrile, 10% ammonium hydroxide 1% triﬂuoroacetic acid, 80% acetonitrile temperature and then were separated by centrifugation (16.1 rcf, 25 C, 10 min). The concentration of troponin-I was measured using the Immulite 1000 Troponin-I System (Siemens Healthcare Diagnostics). Particle eluates were lyophilized overnight, reconstituted in Immulite Sample Diluent (Siemens Healthcare Diagnostics), and assayed accord- ing to the manufacturer’s instructions. Dissociation Constant Calculation. The dissociation constant between the particles and troponin-I was estimated by solving the ordinary differential equations derived from the dissociation equilibrium reaction of bound versus free analyte using the R software, deSolve package.35 LC MS/MS analysis. Eluates from the particles were analyzed by mass spectrometry. Proteins were dried in a SpeedVac and were reconstituted in 8 M urea, reduced by 10 mM DTT, alkylated by 50 mM iodoacetamide, and digested by trypsin (Promega) at 37 C overnight. Tryptic peptides were further purified by Zip-Tip (Millipore) and analyzed by reversed-phase liquid chromatography nanospray tandem mass spectrometry (LC MS/MS) using an LTQ-Orbitrap mass spectrometer (ThermoFisher). After sample injection by auto- sampler, the C18 column (0.2 mm 50 mm, Michrom Bioresources, Inc.) was washed for 2 min with mobile phase A (0.1% formic acid), and peptides were eluted using a linear gradient of 0% mobile phase B (0.1% formic acid, 80% acetonitrile) to 50% mobile phase B in 90 min at 500 nL/min, then to 100% mobile phase B for an additional 5 min. The LTQ mass spectrometer was operated in a data-dependent mode in which each full MS scan was followed by five MS/MS scans where the five most abundant molecular ions were dynamically selected for collision- induced dissociation (CID) using a normalized collision energy of 35%. Tandem mass spectra were searched against the NCBI human database with SEQUEST using tryptic cleavage constraints. High-confidence peptide identifications were obtained by applying the following filter criteria to the search results: Xcorr versus charge g1.9, 2.2, 3.5 for 1+, 2+, 3+ ions; ΔCn > 0.1; probability of randomized identification e0.01. Multiple Reaction Monitoring (MRM) Analysis. Human urine was centrifuged (10 min, 1000 rpm, 4 C) in order to remove the cellular debris, and the specific gravity was recorded. Human growth hormone (hGH) was spiked in human urine at a concentration of 50 ng/mL. Aliquots of 1 mL of urine containing hGH were incubated with 500 μL of Remazol Brilliant Blue R particles for 30 min at room temperature. The particles were separated by centrifugation (16.1 rcf, 10 min, 25 C) and washed with water (16.1 rcf, 10 min, 25 C). Proteins captured by the particles were eluted with 500 μL of elution buffer (70% acetonitrile, 10% ammonium hydroxide) and dried in a vacuum concentrator. Fifty microliters of urine solution and particle eluate were prepared for MRM ARTICLE Figure 4. Light-scattering analysis of particles functionalized with diﬀerent chemical baits. Hydrodynamic diameter (DH) decreased with increases in the temperature of the solution. The temperature diameter relationship is inﬂuenced by the type of dye incorporated. analyses by: (1) reduction with 10 mM dithiothreitol in 1 M urea/ 50 mM ammonium bicarbonate (NH4HCO3) at room temperate for 60 min, (2) alkylation with 50 mM iodoacetamide at room temperature in the dark for 20 min, (3) overnight enzymatic digestion with 0.5 μg of sequencing grade trypsin in 50 mM ammonium bicarbonate, pH 8 at 37 C. Digestions were quenched with the addition of 2 μL of concentrated trifluoroacetic acid and then dried in a vacuum concen- trator. Samples were then reconstituted with 50 μL of 200 μg/mL glucagon (Sigma-Aldrich, St. Louis, MO) in 0.1% formic acid. Sample digests were analyzed with a TSQ Quantum Ultra Triple Quadrupole mass spectrometer (ThermoFisher Scientific). Samples were loaded onto a 1 mm 150 mm Hypersil Gold 3 μm particle C-18 reversed phase column (Thermofisher Scientific) with an Accela autosampler (Thermofisher Scientific) fitted with a 10 μL sample loop. The LC pump was an Accela (ThermoFisher Scientific) operated at 160 μL/min, and effluent was directed into the mass spectrometer using an IonMax source. After sample injection, the column was washed for 5 min with 98% mobile phase A (0.1% formic acid), and peptides were eluted using a linear gradient of 2% mobile phase B (0.1% formic acid in acetonitrile) to 50% mobile phase B in 20 min. The instrument divert valve was switched to waste before and after the peptides eluted in order to keep the source free of excess salts and debris. Both Q1 and Q3 resolution were set to 0.7 fwhm. Q2 contained 1.5 mTorr of argon. The scan width was 0.002 m/z, and dwell time was 15 ms. Collision energies (CE) were calculated according to the formula: 0.034 precursor ion mass m/z + 3.314. Each sample was analyzed in triplicate. Peptide FPTIPLSR (465.77 2+) from hGH, with transitions 392.24, 472.29, 585.37, 686.42, and 783.47 were chosen for MRM analyses on the basis of previous work with tryptic digests of hGH standards. MRM results were loaded into the Pinpoint program, version 1.0 (ThermoFisher Scientiﬁc) for data analysis. Data were divided into two groups: (1) nanoparticle-enriched urine sample (triplicate analyses) and (2) non-nanoparticle urine sample (triplicate analyses). Peak width tolerance was 30 s, the alignment error tolerance was 0.5 min, and the minimum signal threshold was 100. The fragment ion peak areas for all transitions were summed and the average areas calculated using the triplicate analyses for the nanoparticle and non-nanoparticle samples. 19182 dx.doi.org/10.1021/ja207515j |J. Am. Chem. Soc. 2011, 133, 19178–19188