Anal. Chem. 1999, 71, 4727-4733
Analysis for TNF-r Using Solid-Phase Affinity Capture with Radiolabel and MALDI-MS Detection Gregory B. Hurst,*,† Michelle V. Buchanan,†,‡ Linda J. Foote,‡ and Stephen J. Kennel‡
Chemical and Analytical Sciences Division and Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831
Screening of mutant mice for subtle phenotypes requires sensitive, high-throughput analyses of sentinel proteins in functional pathways. The cytokine TNF-r is upregulated during inflammatory reactions associated with autoimmune diseases. We have developed a method to monitor the concentration of TNF-r under physiological conditions. TNF-r is captured, purified, and concentrated using monoclonal antibody-coated microbeads. The capture is efficient (>80%) and can be used in the concentration range 50 ng/mL, as determined by detection of 125I-labeled TNF-r. The bead capture of TNF-r can be combined with direct detection by MALDIMS for sample concentrations of >10 ng/mL. TNF-r can be captured and detected from diluted mouse serum, with minimal interferences observed in the MALDI spectrum. This method is adaptable to high-throughput sample handling with microfluidic devices and automated mass spectrometric analysis. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become an important tool for protein analysis.1,2 Major advantages of this technology include speed, a positive identification of the protein through its mass-to-charge ratio (m/ z), and potential sensitivities at femtomole or lower levels. Biological sources of proteins such as cell homogenates or serum are generally too complex to be analyzed directly by MALDI-MS, and specific proteins in these mixtures must be partially purified before they can be reliably detected.3 One approach to purifying targeted proteins from biological media is affinity separations. Using this approach, peptides and proteins can be captured from biological fluids using antibodies attached to solid surfaces and subsequently detected and partially quantitated using MALDIMS.4-11 These reports have pointed out that the selectivity of * Corresponding author: (phone) (423) 574-7469; (fax) (423) 576-8559; (email)
[email protected]. † Chemical and Analytical Sciences Division. ‡ Life Sciences Division. (1) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A-1203A. (2) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1998, 70, 647R716R. (3) Nelson, R. W.; McLean, M. A.; Hutchens, T. W. Anal. Chem. 1994, 66, 1408-1415. (4) Nelson, R. W.; Krone, J. R.; Bieber, A. L.; Williams, P. Anal. Chem. 1995, 67, 1153-1158. (5) Krone, J. R.; Nelson, R. W.; Dogruel, D.; Williams, P.; Granzow, R. Anal. Biochem. 1997, 244, 124-132. (6) Nelson, R. W.; Krone, J. R.; Jansson, O. Anal. Chem. 1997, 69, 4363-4368. 10.1021/ac9905423 CCC: $18.00 Published on Web 09/11/1999
© 1999 American Chemical Society
affinity separations due to the antibody-antigen interaction is complemented by the m/z measurement capability of mass spectrometry.4-11 For the purposes of screening relatively large numbers of biological samples for the content of targeted proteins, a highthroughput, semiquantitative technique is necessary. We are interested in methods to screen serum samples from mutant mouse populations for the expression of cytokines, which are sentinels of inflammatory reactions. The goal of this work is to detect mutant animals that have relatively subtle abnormalities leading to chronic inflammatory diseases. To this end, we have developed a method for concentration and purification of tumor necrosis factor R (TNF-R), a 17 kDa proinflammatory cytokine, on microbeads that have been covalently derivatized with capture molecules. This method is adaptable to high-throughput sample handling with microfluidic devices and automated mass spectrometric analysis. The data show that TNF-R captured from 100-µL samples can be detected at the 10 ng/mL level using MALDIMS, and the capture methodology extends to lower concentrations, as determined by monitoring 125I-labeled TNF-R. When TNFR-spiked mouse serum samples are subjected to this capture and detection methodology, additional peaks appear in MALDI-MS spectra, but they pose no significant interferences to the TNF-R measurement. Further improvements will be necessary to detect typical physiological concentrations in the picogram per milliliter range. EXPERIMENTAL SECTION Reagents. Purified goat polyclonal antibody to mouse TNF-R (GxTNF-R) was obtained from R&D Systems (Minneapolis, MN), and purified rat monoclonal antibody to mouse TNF-R (RatxTNFR) was obtained from PharMingen (San Diego, CA). Recombinant, carrier-free murine TNF-R was obtained from R&D Systems. A recombinant soluble TNF-R receptor protein, rhu TNFR:Fc, was provided by Immunex Corp. (Seattle, WA). Three solid-phase capture materials were tested. Amino-polystyrene microparticles (0.91-µm-diameter beads) and amino-magnetic microparticles (1.2 µm-diameter beads) were obtained from Spherotech (Libertyville, IL). Sephacryl S-500HR (Amersham Pharmacia Biotech USA, (7) Nelson, R. W.; Krone, J. R.; Jansson, O. Anal. Chem. 1997, 69, 4369-4373. (8) Brockman, A. H.; Orlando, R. Anal. Chem. 1995, 67, 4581-4585. (9) Brockman, A. H.; Orlando, R. Rapid Commun. Mass Spectrom. 1996, 10, 1688-1692. (10) Papac, D. I.; Hoyes, J.; Tomer, K. B. Anal. Chem. 1994, 66, 2609-2613. (11) Liang, X.; Lubman, D. M.; Rossi, D. T.; Nordblom, G. D.; Barksdale, C. M. Anal. Chem. 1998, 70, 498-503.
Analytical Chemistry, Vol. 71, No. 20, October 15, 1999 4727
Piscataway, NJ) was fragmented into ∼1-µm particles by microprobe sonication for 2 min at an output setting of 4 with a Branson Sonifier cell disruptor. Fragmented particles were selected for size on graded nylon mesh filters. MALDI matrix components included sinapinic acid (SA) obtained from Aldrich Chemical Co. (Milwaukee WI) and Immobilon NC Pure nitrocellulose from Millipore Corp. (Bedford MA). Solvents used for MALDI were acetonitrile (DNA synthesis grade, Perkin-Elmer, Foster City, CA) and acetone (HPLC grade, J. T. Baker, Phillipsburg NJ), both used as received, and deionized water obtained from a Millipore purification system with Milli-RO and Milli-Q UV Plus modules. Methods. Spherotech microbeads were treated with 0.5% glutaraldehyde in phosphate-buffered saline (PBS, 0.15 M NaCl, 0.010 M sodium phosphate, pH 7.6) for 5 min and washed twice in PBS.12 Sephacryl particles (50 mg in 100 µL of H2O) were treated for 10 min with 100 µL of a 70 mg/mL solution of CNBr adjusted to pH ∼11.0 by addition of a total of 17 µL of 4 M NaOH.13 One milliter of 0.1 M sodium phosphate (pH 7.6) was added, and the beads were collected by centrifugation and washed once in PBS. Antibody was added at a rate of 5 µg/100 µg of beads and allowed to bind for 3 h at room temperature with gentle mixing. Beads were then treated for 3 h at room temperature in sterile 5 mg/mL bovine serum albumin (BSA) to saturate unbound aldehyde groups and subsequently stored at 4 °C in sterile 5 mg/mL BSA in PBS. Beads were washed twice in PBS before further use. Ten micrograms of TNF-R in carrier-free buffer were radioiodinated with 125I- (Amersham) using Chloramine T oxidation according to published methods.13,14 The labeled protein was diluted into 5 mg/mL BSA carrier protein and purified by gel filtration on a 10-mL open column of Ultragel AcA 44 (Biosepra Inc., Marlborough, MA) equilibrated with 5 mg/mL BSA in PBS. Fractions containing labeled protein were pooled and stored frozen at -20 °C until used. For capturing the analyte from solution, a suspension of antibody-derivatized microparticles was mixed with varying amounts of TNF-R in a 100-µL volume of 5 mg/mL BSA in PBS in 0.2-mL polypropylene tubes (Costar, Corning, Corning, NY) and incubated with end-over-end tube rotation at room temperature for 3 h. In experiments testing the effects of mouse serum, normal serum from BALB/c mice was included with the TNF-R sample. The normal serum used was tested by ELISA assay and found to contain 10 from different locations on the sample spot.
captured on polystyrene beads derivatized with RatxTNF-R. The signals are quite intense for the 5- and 30-ng samples, originally in 0.1-mL volumes. For the 1-ng sample, corresponding to an original sample concentration of ∼0.6 nM, the signal is detectable, but the S/N is
