Comparison of Ovalbumin Quantification Using Forward-Phase

The data obtained from the forward-phase protein arrays gave values that ... Monoclonal Antibody Selection for Interleukin-4 Quantification Using Susp...
0 downloads 0 Views 181KB Size
Download PDF
Comparison of Ovalbumin Quantification Using Forward-Phase Protein Microarrays and Suspension Arrays L. Wang,*,† K. D. Cole,† Hua-Jun He,† D. K. Hancock,† A. K. Gaigalas,† and Y. Zong‡ Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8312, and Full Moon BioSystems, Inc., 754 N. Pastoria Avenue, Sunnyvale, California 94085 Received March 2, 2006

We employed ovalbumin (a simulant used for ricin and botulism toxins in biodefense applications) and its high affinity polyclonal antibody as a model system to examine the sensitivity, dynamic range, linearity, and reproducibility of forward-phase array results in comparison to suspension arrays. It was found that protein microarrays had a dynamic range of 4 orders of magnitude and a sensitivity of less than 1 pg/mL, respectively. The dynamic range and sensitivity of suspension arrays were close to 2 orders of magnitude and 0.25 ng/mL, respectively. The sensitivity we observed for the suspension arrays is comparable to that reported for enzyme-linked immunosorbent assays (ELISAs) in the literature. We used ovalbumin samples with two different purities, 38.0% and 76.0% (w/w), as determined by polyacrylamide gel electrophoresis (PAGE). These samples were used to evaluate the effect of impure samples on detection. The data obtained from the forward-phase protein arrays gave values that were consistent with the PAGE data. The data from the suspension arrays were not as consistent and may indicate that this format may not give as reliable data with impure samples. Knowledge of the advantages and disadvantages of the two proteomic methods would allow their more rational use in clinical diagnosis. Keywords: ovalbumin • quantification • forward-phase protein microarrays • suspension arrays • sensitivity • dynamic range • linearity • reproducibility • accuracy

Introduction After the completion of the human genome sequencing project, DNA microarrays and sophisticated bioinformatics tools give researchers a global view of biological systems. Efforts are undertaken to adapt microarray methodology to analyze protein expressions and protein-protein interactions in a massively parallel mode in proteome era.1,2 The growing demands of genomics and proteomics for the analysis of gene and protein function in a global perspective have drastically increased interest in microarray-based bioassays.3-5 For protein expression profiling, current technologies, such as two-dimensional gel electrophoresis in combination with mass spectrometry, allow the identification of biologically relevant proteins with a high resolving power, but have considerable limitations, i.e., low abundance proteins are poorly represented.6 Yet, protein microarrays provide an alternative for the detection of low-expressed proteins in highthroughput fashions. Two formats exist including forward- and reverse-phase protein microarrays that depend on the type of bait molecules immobilized on the solid substrates, either antibodies in the former or analyte proteins in the latter.7,8 Although there are publications in thousands concerning the use of protein microarrays, the technology is still in its infancy. Many issues, such as antibody affinity, sample conservation, * To whom correspondence should be addressed. National Institute of Standards and Technology, 100 Bureau Drive, Stop 8312, Gaithersburg, MD 20899-8312, Voice: (301) 975-2447. E-mail: [email protected]. † National Institute of Standards and Technology. ‡ Full Moon BioSystems, Inc.

1770

Journal of Proteome Research 2006, 5, 1770-1775

Published on Web 06/21/2006

labeling and amplification chemistries, and quantification need to be resolved or improved before protein microarrays can be rationally used in clinical diagnosis.9-11 At the present, the planar protein microarray approaches have not achieved full automation. Bead-based arrays are an interesting alternative to planar microarrays; particularly when the number of components to be measured in parallel is relatively small.12,13 The latest designs of the suspension array flow cytometer (Luminex 100 from Luminex Corporation and BioPlex from BioRad) provide high sensitivity, high throughput, and multiplexed detection at a low cost. The instrument is designed to take advantage R-phycoerythrin (PE), a highly fluorescent protein, as the reporter. These arrays utilize polystyrene microbeads (5.5 µm in diameter) that are impregnated with precise ratios of red and infrared fluorescent classification dyes yielding an array of 100 bead sets, each with a unique intensity address. A 635 nm, 10 mW red diode laser excites the two classification fluorophores embedded in the microbeads, and a 532 nm, 13 mW doubled yttrium aluminum garnet (YAG) laser provides the excitation of the fluorescent reporter PE. The immunoassays adopt a typical sandwich immunoassay format where antigen-specific capture antibodies are immobilized on the microbeads, antigen is then added and allowed to bind to capture beads, and the bound antigen is subsequently recognized by the biotinylated secondary antibodies that are identified by streptavidin-PE. Because the reporter fluorescence does not overlap with the classification signal, no fluorescent compensation is required. Sensitivity and reproducibility of the suspension arrays are similar to those observed with wellestablished enzyme-linked immunosorbent assays (ELISAs).12,14 10.1021/pr060074v CCC: $33.50

 2006 American Chemical Society

Comparison of Ovalbumin Quantification

Figure 1. Illustrations of the forward-phase protein microarrays (A) and suspension arrays (B). In forward-phase microarrays, capture antibodies are printed on planar substrates and used to bind analyses of interest that are further identified by labeled secondary antibodies. Three components form a sandwich complex whose fluorescence signal is measured by using a microarray scanner. In suspension arrays, capture antibodies are immobilized on Luminex microbead surfaces and used to capture analytes. The bound analytes are subsequently recognized by biotinylated secondary antibodies that are identified by streptavidin-phycoerythrin conjugate. The fluorescence signals from formed complexes on bead surfaces are quantified by using a Luminex flow cytometer.

Quantitative protein analyses using various proteomic methodologies are mostly characterized by the sensitivity and dynamic range of the detection. The accuracy and specificity of the assays are rarely discussed due to the lack of protein standards. Additionally, there are a limited number of comparative studies focusing on the quantification aspects of the proteomic methods based on the affinity binding mechanisms. In this study, we employed ovalbumin (a simulant commonly used for ricin and botulism toxins in biodefense applications) and its high affinity polyclonal antibody as a model system to examine the sensitivity, reproducibility, dynamic range, and linearity of forward-phase array results with respect to suspension arrays. Conventional sandwich-type immunoassays were implemented in the two array platforms (Figure 1). The accuracy and specificity of these two proteomic methods were evaluated by using two analyte protein samples with different ovalbumin purities.

Material and Methods Two albumin samples from chicken egg white, Grade V (A5503) and Grade II (A5253), were both purchased from Sigma Chemical Company (St. Louis, MO) and used as supplied. (Certain commercial equipment, instruments, and materials are identified in this paper to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment are necessarily the best available for the purpose.) An affinity-purified rabbit anti-ovalbumin (1.0 mg/mL in PBS, pH 7.2, 0.1% sodium azide) was obtained from Immunology Consultants Laboratory, Inc. (Newberg, OR). Mono-reactive Cy3 dye packs were from Amersham Biosciences (Piscataway, NJ).

research articles FMB protein printing buffer and FMB protein slides are products from Full Moon BioSystems (Sunnyvale, CA). Ovalbumin Purity Determination. Two protein samples were gently dissolved in PBS pH 7.4 and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using the NuPAGE Novex Bis-Tris System (Invitrogen, Carlsbad, CA). The multiple diluted samples were mixed with the sample buffer with a reducing agent and heated at 70 °C for 10 min. These samples were then run on 1 mm polyacrylamide gels (4% to 12%) using the 3-(N-morpholino)-propanesulfonic acid (MOPS) buffer system at 200 V for 50 min. Prestained molecular weight protein standards were also run on the gel (broad range 6 to 175 kDa, New England BioLabs, Ipswich, MA). Gels were stained using SimplyBlue (Invitrogen, Carlsbad, CA) microwave procedure according to the manufacturer’s recommendations. Gels were then imaged on a white light illuminator and the images saved as 12-bit TIF files. The fluorescence bands were first identified by using commercial software (Image-Gel-Pro, version 3.1, Media Cybernetics, Silver Springs, MD), and then the number of bands for the same protein sample (Grade II or V) was kept the same across various concentration preparations. The fluorescence intensity of each band was obtained, and the ovalbumin purity was calculated as the ratio of the intensity of the ovalbumin band to the sum of the intensities of all the bands. Antibody Specificity. Two albumin samples from chicken egg white, Grade V (3 µg) and Grade II (10 µg), were loaded in separate wells in an agarose gel and electrophoresed at 200 V for 50 min. The affinity-purified rabbit anti-chicken ovalbumin (1 mg/mL) was then added to the trough between the two protein loading wells and incubated overnight before staining with Comassie brilliant blue R 250 (Sigma, St. Louis, MO) and subsequent imaging. Labeling Antibody with Cy3 Fluorophores. The rabbit antiovalbumin antibodies in PBS went through buffer exchange to 0.1 M sodium carbonate buffer, pH 9.3, by using Pro Spin columns from Princeton Separations, Inc. (Adelphia, NJ). Conjugation of Cy3 fluorophores to the antibodies was carried out according to the manufacturer-recommended procedure. The same spin columns were used for separation of the antibodies from free fluorophores and buffer exchange to PBS, pH 7.2, 0.05% sodium azide. The fluorophore per antibody ratio was estimated to be ∼six based on the absorbance at 552 nm and at 280 nm. Forward-Phase Protein Microarray Measurements and Retention Determination. The affinity-purified polyclonal antibodies were diluted with FMB protein printing buffer in consecutive 2-fold dilutions from 500 µg/mL to 0.98 µg/mL. These solutions were spotted on FMB protein slides, each with 8 replicates, using Amersham Gen III spotter. After printing, the slides were kept in 65% to 70% humidity chamber for 10 h and then dried for 30 min at room temperature. The slides were blocked with 1× PBS/1% BSA, and then washed briefly with Tris buffer saline (TBS, 10 mM Tris-HCl, pH 7.5, 150 mM NaCl)/ 0.1% Tween 20. After rinsed with deionized water, the slides were dried with a stream of 25 psi nitrogen. The reaction with analytes was performed for 1 h in Whatman 16-well incubation chambers equipped with a chip clip slide holder (50 µL protein antigen solution per well). After washed with TBS with Tween 20 (TBS/0.1% Tween 20) for 30 min at room temperature and subsequently rinsed with deionized water, the slides were dried with a gentle stream of nitrogen. The coupling of Cy3-labeled antibodies was also performed in the 16-well incubation Journal of Proteome Research • Vol. 5, No. 7, 2006 1771

research articles chambers equipped with a chip clip slide holder for 1 h. After washed with TBST (TBS/0.1 % Tween 20) for 30 min at room temperature and subsequently rinsed with deionized water, the slides were dried with a stream of nitrogen and ready for fluorescence measurements by using an Axon scanner, GenePix 4000 (Molecular Devices, Sunnyvale, CA). For each array spot, a signal-to-noise ratio (SNR) is calculated as the mean fluorescence intensity with the mean background intensity subtracted divided by the standard deviation of the background signals.15,16 To assess retention values, Cy3-labeled antibodies were diluted with the printing buffer in nine consecutive 2-fold dilutions from 100 µg/mL to 0.20 µg/mL and spotted on FMB protein slides. After humidity treatment, the printed slides were washed with TBS, rinsed with deionized water, and dried with a gentle stream of nitrogen. The first fluorescence reading was performed. These slides then went through coupling and washing procedures described above in the absence of an analyte and antibody, so-called ‘Mock Coupling’. After washing and rinsing procedures, the slides were dried with a stream of nitrogen, and the second fluorescence reading was carried out. The retention values were determined by the ratio of the signal after mock coupling and washing (the second fluorescence reading in terms of SNR) and signal after printing (the first reading in terms of SNR). Suspension Array Detections. The immobilization of the rabbit anti-ovalbumin antibody to Luminex carboxylated microspheres was accomplished through formation of a carbodiimide bond and carried out according to the manufacturerrecommended procedures. A FluoReporter Mini-Biotin-XXProtein Labeling Kit from Molecular Probes was used for biotinylation of the polyclonal antibody, and the coupling reaction of biotins to the antibody was carried out according to the manufacturer-recommended procedures. A commercial Luminex 100 instrument (Luminex Corp, Austin, TX) was used to perform the multiplexing sandwich assays detecting ovalbumin antigen. Polyclonal antibody coupled to specific Luminex beads (1000-1500 beads, 14 µL) were used for the primary analyte-capture step. After incubation with analytes (20 µL) for 20 min, the biotin-labeled antibodies (30 µL, 0.8 µg) were added and incubated for 20 min to form the sandwichtype complexes. PE-labeled streptavidin (10 µL, 1.5 µg) was then added as the fluorescent reporter for ovalbumin detection. The blocking buffer, pH 7.4, (0.01 M phosphate, 0.138 M NaCl, 0.0027 M KCl, 1% (w/w) bovine serum albumin, 0.05% (w/w) NaN3) was used to make stock solutions for the sandwich assays in 96 well plates (Model P, Corning Incorporated, Corning, NY). For data analyses, the blank signals were obtained in the absence of analytes and were subtracted from the fluorescence signals of the sandwich complexes in the presence of analytes.

Results The ovalbumin purities in the two albumin samples, Grade V and II, were determined by using SDS-PAGE. On the basis of the gel image obtained for multiple diluted albumin samples shown in Figure S1 in the section of ‘Supporting Information’, the ovalbumin purities were determined to be 76.0% (s.d., 3.5%) and 38.0% (s.d., 2.5%) for albumin Grade V and II, respectively. The ratio of ovalbumin contents between grade II protein sample and grade V sample is therefore 0.5. To verify the specificity of the commercially available, affinity purified rabbit anti-ovalbumin antibody, we carried out immuno-electrophoreses of the two ovalbumin samples against 1772

Journal of Proteome Research • Vol. 5, No. 7, 2006

Wang et al.

Figure 2. Standard curves for albumin (Grade V) plotted as signal-to-noise ratio (SNR) as functions of albumin concentration and antibody printing concentration. The antibody concentrations (µg/mL) are as follows: 0.98 (9), 1.95 (b), 3.91(2), 7.81 (1), 15.63 ([), 31.25 (×), 62.5 (/), 125 (O), 250 (4), 500 (]). The rabbit antiovalbumin antibodies labeled with Cy3 fluorphores as the reporter were kept in excess (2.5 µg/mL). The solid line shows the fit of SNR at the antibody printing concentration of 62.5 µg/ mL to the logistic model described in the text. The inset exhibits the dependence of the parameter of the fit (SNRmax) on the printing concentration (pc).

the antibody, shown in Figure 2S in the section of ‘Supporting Information’. The two symmetric arcs against the antibodyloading trough confirmed that the antibody is specific to ovalbumin. It is known that with an array slide, the type of bait molecules to be printed and the printing buffers will greatly affect the array data quality. For the forward-phase protein array study, we used Cy3-labeled anti-ovalbumin antibody to assess the retention values of the antibody at various printing concentrations, which are given in Figure S3 in the section of ‘Supporting Information’. These values increase monotonically until reaching a plateau. Under the saturation condition, close to 40% of antibody molecules was retained on each array spot. We attempted to fit the measured retention values (r) at different antibody printing concentrations (pc) to an exponential function given by r(pc) ) r0 + a(1 - e-k*pc) Here r0 is the minimum value of r, a is the amplitude of the exponentially increasing part, and k is the rate of increase. The continuous line is the fitting curve by the exponential function (Figure S3). From 1 µg/mL to about 20 µg/mL the increase of the retention factor is exponential. This suggests that the retention of a given antibody is enhanced by the presence of other antibodies on the surface. At a concentration of 9.1 µg/ mL, half of the maximum retention is achieved. The saturation of the retention value suggests that the antibody has to interact with the surface, and that there is a maximum number of such interaction sites. The exponential dependence on printing concentration suggests the presence of cooperative interaction among the antibodies. At lower concentrations, the retention factor lies below the values given by the exponential increase model. This suggests that at lower concentrations the rate of retention may be proportionate to the number of interaction sites on the surface resulting in a retention which depends linearly on the antibody concentration in the printing solution.

research articles

Comparison of Ovalbumin Quantification

A diagram of ovalbumin detection using the forward-phase protein arrays is shown in Figure 1A. While the rabbit antiovalbumin antibody labeled with Cy3 fluorophores was kept in excess (2.5 µg/mL, 45 µL/well), the standard curves generated for albumin protein (Grade V) were plotted as functions of albumin concentration and antibody printing concentration and displayed in Figure 2. When albumin concentration is below 16 ng/mL, SNR increases monotonically as increasing the protein concentration. The linearity range of the detection spans close to 3 orders of magnitude and the dynamic range extends over 4 orders of magnitude. The detection sensitivity of the protein array assays is as low as 1 pg/mL. At protein concentrations higher than 16 ng/mL, the signal reaches saturation under the present photomultiplier tube (PMT) settings of the scanner. The continuous line in Figure 2 shows a fit of SNR at a printing concentration of 62.5 µg/mL (/ symbols) to a logistic model given by SNR ) SNRmax +

(SNRmin - SNRmax) C 1+ Cmid

where SNRmin is the minimum value and SNRmax is the maximum value of SNR respectively, and Cmid is the value of the albumin concentration, C, at which SNR is half of its maximum value.17 The fit gives a good representation of the data except at the lowest value of the albumin concentration. Similar fits were obtained at other printing concentrations. The inset of Figure 2 shows the dependence of the parameter of the fit on the printing concentration, pc. As expected, SNRmax, increases with the printing concentration. The increase is a quadratic function of the printing concentration, and beyond 200 µg/mL the value of SNRmax starts to level off. The interpretation of the parameter SNRmin is complicated by the poor fit at lower values of the printing concentration revealing the limitation of the simple logistic model. We further performed protein array measurements for the grade II ovalbumin sample. SNR as a function of protein concentration is shown at two antibody printing concentrations in Figure 3A for the two albumin samples with different ovalbumin purities. With the same protein concentration (ng/ mL), the signal from grade II albumin sample is consistently lower than that of grade V sample due to lower ovalbumin purity in the sample. The ratio of SNR from grade II protein sample to that of grade V sample is calculated and displayed in Figure 3B as a function of the protein concentration. These values are scattered between 0.4 and 0.54 with a mean value of 0.47. The error associated with the ratios determined by using protein microarrays with respect to the value by PAGE (0.5) is found to be -6%. Suspension array measurements were performed on the samples with different ovalbumin purities. The antibody sandwich complexes formed (Figure 1B) are similar in structure to those in protein array assays except that the secondary antibody is biotinylated rabbit anti-ovalbumin antibody, and subsequent binding with PE-labeled streptavidin is required for the identification of the formed sandwich complexes. Figure 4 shows standard curves generated for the two protein samples at various concentrations (albumin grade II, solid circle; grade V, open circle). The standard deviations are less than 10%. The inset displays the ratios of ovalbumin purities obtained at various analyte concentrations. The mean value of these ratios

Figure 3. (A) Signal-to-noise ratio (SNR) as a function of albumin concentration at two antibody-printing concentrations (125 µg/ mL and 250 µg/mL), blue and red bars for grade V albumin sample, and yellow and green bars for grade II albumin protein. (B) Ratio of ovalbumin purities in the two samples calculated as the ratio of SNR from grade II albumin sample to that from grade V protein sample as a function of protein concentration at two antibody-printing concentrations, 125 µg/mL (solid circle) and 250 µg/mL (star /). The dotted line refers to the ratio value (0.50) determined by PAGE.

is 0.78, which is higher than the value determined by PAGE (0.5) and forward-phase protein arrays (0.47, Figure 3B). A 56% error is calculated with regard to the ratio value determined by PAGE.

Discussion The solid line fitting by using the logistic model in Figure 2 gives a reasonably good representation of the data. Similar fits were also obtained at other antibody printing concentrations. In all cases, the measured value of SNR at the lowest concentration of albumin was smaller than expected from the fit to the model. The SNR at the lowest albumin concentrations exhibits a more linear dependence on the concentration of albumin and does not approach a SNRmin that represents some inherent background response of the instrument. A single site saturation model with no instrument background response, gives a linear dependence of SNR at low albumin concentration but a poor fit over the entire concentration range. It is not clear how to model the SNR at low albumin concentrations. Unlike SNRmax whose value is almost independent of the model used Journal of Proteome Research • Vol. 5, No. 7, 2006 1773

research articles

Figure 4. Fluorescence intensity as a function of analyte protein concentration generated by using suspension arrays for the two albumin proteins, albumin grade II (solid circle) and grade V (open circle). The error bar refers for the standard deviation of each value averaged from replicates in the same experiment and from different experiments. The inset shows the ratios of ovalbumin purities between grade II albumin and grade V protein obtained at different analyte protein concentrations. The dash line refers to the ratio value (0.50) determined by PAGE.

to fit the data, the interpretation of the parameters, SNRmin and Cmid, is not clear. The forward-phase protein microarrays and suspension arrays are both suitable for high-throughput protein expression profiling and protein function studies. Although the same type of sandwich protein assays are implemented in the present study (Figure 1), distinct differences between the two-proteomic methods attribute to the outcome of the ovalbumin quantification. The protein array assays are several hundred times more sensitive than the suspension arrays. The detection dynamic range of the protein microarrays is approximately 2 orders of magnitude better than that of suspension arrays (Figure 2 and Figure 4). The protein slides used in this study contain mainly epoxy, aldehyde, and hydroxyl functional groups on surfaces allowing covalent attachment of antibody molecules. When 1 nL of the antibody stock solution at a concentration of 500 µg/mL is printed on an array spot with a diameter of 250 µm, the antibody density on a spot is 5 × 10-6 ng/µm2 assuming a 50% retention value (Fig. S3) for the antibody. In contrast, a 5.6 µm Luminex bead has a surface area of approximately 95 µm2. Assuming a 80% coupling efficiency for antibodies conjugated to carboxylated bead surfaces, 50 µg of rabbit anti-ovalbumin antibody was used for coupling of 2.5 × 106 beads yielding an antibody density on bead surface of 1.7 × 10-4 ng/µm2. On the basis of these approximations, the antibody density for suspension array measurements is about 30 times more than that for forwardphase protein microarrays. This suggests that in theory suspension array results could be more sensitive than that of protein arrays because of higher density of the capture antibody. In addition, different reporting fluorophores are utilized in the two array platforms. In suspension arrays, PE conjugated streptavidin served as the fluorescence report following the affinity binding with biotinylated antibodies. It is known that a single PE molecule is composed of 30 fluorophores (phycoerythrobilin and phycourobilin) and has a high fluorescence quantum yield.18 This fluorophore has a molecular weight of 1774

Journal of Proteome Research • Vol. 5, No. 7, 2006

Wang et al.

243 kDa and is about twice as large as an antibody while attached to streptavidin (52 kDa). The large size of streptavidinPE conjugate and the hydrophobicity of these proteins in the absence of a washing step might contribute to high fluorescence background for the suspension array assays that affects both assay sensitivity and limit of detection.19 On the other hand, the use of Cy3-labeled rabbit anti-ovalbumin antibodies in forward-phase protein arrays, incubation chambers instead of cover slides for enhancing the efficiencies of the affinity binding interactions, and washing steps after each affinity binding reaction result in low fluorescence background. With proper use of an array scanner,16,20 a linearity range of about 3 orders of magnitude can be readily accomplished. Vastly different detection methodologies are employed in the two proteomic platforms. In the present study, a nonconfocal microarray scanner was used for fluorescence measurements of protein array spots. This scanner is capable of detecting two to four Cy3 fluorophores per µm2.16 A typical flow cytometer has a limit of detection of 12 fluorescein molecules per µm2.21,22 Since the products of the maximal absorptivity and fluorescence quantum yield are similar for fluorescein and Cy3 fluorophore, it is logical to hypothesize that the detection limit of suspension array instruments is about 12 Cy3 fluorophores per µm2. Since a single PE molecule has 30 fluorophores (phycoerythrobilin and phycourobilin), in principle, the suspension array instrument may be capable of measuring less than a single PE per µm2 (personal communication, Robert Hoffman, BD Biosciences). With the use of suspension arrays, the dynamic range of ovalbumin detection is about 2 orders of magnitude (Figure 4), and the sandwich assays are able to measure 0.25 ng/mL of ovalbumin protein. These results are comparable to those reported by McBride and co-workers.14 These authors also confirmed their suspension array results with those obtained by using conventional ELISAs. Nevertheless, we have found in the present study that suspension arrays may not give as reliable data with impure samples as forward-phase protein arrays. The mean value of ovalbumin purity ratios (0.78, Figure 4) is higher than the value determined by PAGE (0.5) and forward-phase protein arrays (0.47, Figure 3B). The error associated with the ratio values (56%) is much larger than that by using forward-phase protein arrays (-6%). Although suspension arrays have the potential to be highly automated and miniaturized, care is needed to ensure the accuracy of the technology and to avoid false positive results. We demonstrated in this study that by using forward-phase protein microarrays, we were able to detect less than 1 pg/mL of ovalbumin while taking into account of ovalbumin purity in grade V albumin sample (Figure 2). In other words, the sensitivity for ovalbumin detection is close to 1 × 10-14 M, which is highly desirable with respect to other methods.11 With the use of alternative signal amplification methods, such as a rolling-circle amplification,23 proximity-dependent DNA ligation assays,24 protein-DNA fusions,25 or nanometer microspherebased methods,26 it is likely that the sensitivity of the protein array assays can be further improved to detect sub-femtomolar protein quantities.

Conclusion In the expanding field of proteomics, it is important to be able to separate complex protein mixtures with increasing sensitivity, reproducibility, accuracy, and automation. We employed ovalbumin protein and its high affinity polyclonal

research articles

Comparison of Ovalbumin Quantification

antibody as a model system to examine the reproducibility, sensitivity, dynamic range, and accuracy of the forward-phase array results in comparison to the suspension arrays. It was found that protein microarrays had a dynamic range of 4 orders of magnitude and a sensitivity of about 0.5 pg/mL, respectively. The dynamic range and sensitivity of suspension arrays were somewhat less, approximately 2 orders of magnitude and 0.25 ng/mL, respectively. The sensitivity we observed for the suspension arrays is comparable to that reported for ELISAtype assays in the literature. The use of two albumin samples with different ovalbumin purities, 38.0% and 76.0% (w/w), as determined by SDS-PAGE allows examining the accuracy of the two proteomic methodologies. The data obtained from the forward-phase protein arrays gave values that were consistent with the PAGE data. The data from the suspension arrays was not as consistent and may indicate this format may not give as reliable data with impure samples. The present study suggests that proper controls, such as two different levels of a known protein, can be used not only for adjusting slide-toslide and/or experiment-to-experiment variations, but also for ensuring assay accuracy. Sensitivity, reproducibility, and accuracy are the prerequisites for the rational use of these proteomic methods in clinical diagnosis.

Acknowledgment. The authors are indebted to Dr. John Leslie at Immunology Consultants Laboratory, Inc. for running immuno gel electrophoresis of two albumin samples and obtaining the gel image for the present work. Supporting Information Available: The image of SDS-PAGE of multiple diluted protein samples is shown in Figure S1. Figure S2 displays the immuno-electrophoreses of two albumin samples for verifying the antibody specificity. The retention values determined through mock hybridization are plotted against the printing concentrations of Cy3-labeled antibodies in Figure S3. An exponential fit of the measured retention values at different antibody printing concentrations is also shown in Fig. S3. This material is available free of charge at http://pubs.acs.org. References (1) MacBeath, G. Protein microarrays and proteomics. Nat. Genet. 2002, 32 (Suppl.), 526-532. (2) Templin, M. F.; Stoll, D.; Schwenk, J. M.; Potz, O.; Kramer, S.; Joos, T. O. Protein microarrays: promising tools for proteomic research. Proteomics 2003, 3, 2155-2166. (3) Washburn, M. P.; Koller, A.; Oshiro, G.; Ulaszek, R. R.; Plouffe, D.; Deciu, C.; Winzeler, E.; Yates, J. R. Protein pathway and complex clustering of correlated mRNA and protein expression analyses in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 2003, 100, 3107-3112. (4) Nishizuka, S.; Chen, Sing-Tsung, Gwadry, F. G.; Alexander, J.; Major, S. M.; Scherf, U.; Reinhold: W. C.; Waltham, M.; Charboneau, L.; Young, L.; Bussey, K. J.; Kim, S.; Lababidi, S.; Lee, J. K.; Pittaluga, S.; Scudiero, D. A.; Sausville, E. A.; Munson, P. J.; Petricoin III, E. F.; Liotta, L. A.; Hewitt, S. M.; Raffeld, M.; Weinstein, J. N. Diagnostic Markers that distinguish colon and ovarian adenocarcinomas: Identification by genomic, proteomic, and tissue profiling. Cancer Res. 2003, 63, 5243-5250. (5) Bartling, B.; Hofmann, H.-S.; Boettger, T.; Hansen, G.; Burdach, S.; Silber, R.-E.; Simm, A. Comparative application of antibody and gene array for expression profiling in human squamous cell lung carcinoma. Lung Cancer 2005, 49, 145-154. (6) Gygi, S. P.; Corthals, G. L.; Zhang, Y.; Rochon, Y.; Aebersold, R. Evalution of two-dimensional gel electrophoresis-based proteome analysis technology. Proc. Natl. Acad. Sci. USA 2000, 97, 93909395. (7) Miller, J. C.; Zhou, H.; Kwekel, J.; Cavallo, R.; Burke, J.; Butler, E. B.; Teh, B. S.; Haab, B. B. Antibody microarray profiling of human prostate cancer sera: Antibody screening and identification of potential biomarkers. Proteomics 2003, 3, 56-63.

(8) Paweletz, C. P.; Charboneau, L.; Bichsel, V. E.; Simone, N. L.; Chen, T.; Gillespie, J. W.; Emmert-Buck, M. R.; Roth, M. J.; Petricoin III, E. F.; Liotta, L. A. Reserse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front. Oncogene 2001, 20, 19811989. (9) Liotta, L. A.; Espina, V.; Mehta, A. I.; Calvert, V.; Rosenblatt, K.; Geho, D.; Munson, P. T.; Young, L.; Wulfkuhle, J.; Petricoin, E. F.; III. Protein microarrays: Meeting analytical challenges for clinical applications. Cancer Cell 2003, 3, 317-325. (10) Barry, R.; Soloviev, M. Quantitative protein profiling using antibody arrays. Proteomics 2004, 4, 3717-3726. (11) Poetz, O.; Schwenk, J. M.; Kramer, S.; Stoll, D.; Templin, M. F.; Joos, T. O. Protein microarrays: catching the proteome. Mechanisms of Aging and Development 2005, 126, 161-170. (12) Kellar, K. L.; Iannone, M. A. Multiplexed Microsphere-based Flow Cytometric Assays. Exp. Hematol. 2002, 30, 1227-1237. (13) de Jager, W.; te Velthuis, H.; Prakken, B. J.; Kuis, W.; Rijkers, G. T. Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells. Clin. Diagn. Lab. Immunol. 2003, 10, 133-139. (14) McBride, M. T.; Gammon, S.; Pitesky, M.; O’Brien, T. W.; Smith, T.; Aldrich, J.; Langlois, R. G.; Colston, B.; Venkateswaran, K. S. Multiplexed liquid arrays for simultaneous detection of simulants of biological warfare agents. Anal. Chem. 2003, 75, 1924-1930. (15) Worley, J.; Bechtol, K.; Penn, S.; Roach, D.; Hanzel, D.; Trounstine, M.; Barker, D. A systems approach to fabricating and analyzing DNA microarrays. In Microarray Biochip Technology; Schena, M., Ed.; Eaton Publishing: 2000; Chapter 4, pp 65-85. (16) Zong, Y.; Wang, Y.; Zhang, S.; Shi, Y. How to evaluate a microarray scanner. In Microarrays Methods and Applications-Nuts & Bolts; Hardiman, G. Ed.; DNA Press: 2003; Chapter 5, pp 99-114. (17) Christopoulos, T. K.; Diamandis, E. P. The theory of immunoassays. In Immunoassays; Diamandis, E. P., Christopoulos, T. K., Ed.; Academic Press: New York, 1996; Chapter 3, pp 25-50. (18) Gaigalas, A. K.; Gallagher, T.; Cole, K. D.; Singh, T.; Wang, L. A multistate model for the fluorescence response of R-phycoerythrin. Photochem. Photobiol. 2006, in press, (published on web, www.aspjournal.com, on January 18, 2006). (19) Rao, R. S.; Visuri, S. R.; McBride, M. T.; Albala, J. S.; Matthews, D. L.; Coleman, M. A. Comparison of Multiplexed Technologies for Detection of Bacterial and Viral Proteins. J. Proteome Res. 2004, 3, 736-742. (20) Shi, L.; Tong, W.; Su, Z.; Han, T.; Han, J.; Puri, R. K.; Fang, H.; Frueh, F. W.; Goodsaid, F. M.; Guo, L.; Branham, W. S.; Chen, J. J.; Xu, Z. A.; Harris, S. C.; Hong, H.; Xie, Q.; Perkins, R. G.; Fuscoe, J. C. Microarray scanner calibration curves: characteristics and implications. BMC Bioinformatics 2005, 6(Suppl. 2), S11. (21) Report of Investigation of RM 8640 (microspheres with immobilized fluorescein isothiocyanate) issued on February 19, 2004 by NIST. The certificate includes the MESF values of the reference beads and the uncertainties of the assignments. (22) Gaigalas, A. K.; Wang, L.; Schwartz, A.; Marti, G. E.; Vogt, R. F. Quantitating fluorescence intensity from fluorophore: Assignment of MESF values. J. Res. Natl. Inst. Stand. Technol. 2005, 110, 101-114. (23) Schweitzer, B.; Roberts, S.; Grimwade, B.; Shao, W.; Wang, M.; Fu, Q.; Shu, Q.; Laroche, I.; Zhou, Z.; Tchernev, V. T.; Christiansen, J.; Velleca, M.; Kingsmore, S. F. Multiplexed protein profiling on microarrays by rolling-circle amplification. Nat. Biotechnol. 2002, 20, 359-365. (24) Fredriksson, S.; Gullberg, M.; Jarvius, J.; Olsson, C.; Pietras, K.; Gustafsdottir, S. M.; Ostman, A.; Landegren, U. Protein detection using proximity-dependent DNA ligation assays. Nat. Biotechnol. 2002, 20, 473-477. (25) Weng, S.; Gu, K.; Hammond, P. W.; Lohse, P.; Rise, C.; Wagner, R. W.; Wright, M. C.; Kuimelis, R. G. Generating addressable protein microarrays with PROfusion covalent mRNA-protein fusion technology. Proteomics 2002, 2, 48-57. (26) Wang, L.; Cole, K. D.; Gaigalas, A. K.; Zhang, Y.-Z. Fluorescent nanometer microspheres as a reporter for sensitive detection of simulants of biological threats using multiplexed suspension arrays. Bioconjugate Chem. 2005, 16, 194-199.

PR060074V Journal of Proteome Research • Vol. 5, No. 7, 2006 1775