Determination of Relative Protein Abundance by Internally Normalized

Min-Kyu Han , Young-Hee Oh , Jimin Kang , Young-Pil Kim , Soowon Seo , Jhingook Kim ... Expert Review of Molecular Diagnostics 2007 7 (5), 673-686 ...
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Determination of Relative Protein Abundance by Internally Normalized Ratio Algorithm with Antibody Arrays Oskar Andersson, Mark Kozlowski, Tatiana Garachtchenko, Corina Nikoloff, Nancy Lew, David J. Litman, and Grigoriy Chaga* BD Biosciences Clontech, 1020 E. Meadow Circle, Palo Alto, California 94303 Received December 1, 2004

In this paper, we report an experimental setup and mathematical algorithm for determination of relative protein abundance from directly labeled native protein samples applied to an array of antibodies. The application of the proposed experimental system compensates internally at each array element for a number of deficiencies in array experiments such as differential labeling efficiency in dual color assay systems, differential solubility of protein molecules in dual color assay systems, and differential affinity of capture reagents toward proteins labeled with two different fluorescent dyes. This system offers full compensation for variable amounts of capture reagents on separate array structures, as well as limited compensation for nonspecific interactions between capture reagents and analytes. The proposed experimental strategy enables the use of a large number of capture reagents to develop a true multiplex analysis system that will yield complete relative protein abundance information in two biological systems. Keywords: antibody array • dual color detection • protein abundance • multiplex protein analyses • internally normalized ratio

1. Introduction The completion of the main body of work on the elucidation of the Human Genome has generated a strong need for novel, high-throughput analysis tools and systems that can determine the abundance, post-translational modifications, and biological function of the Proteome encoded by the Human Genome. An increasing number of publications cover the topic of the application of antibodies as capture and/or detection reagents in high-throughput analyses of protein abundance.1-9 Strong development in the field of DNA arrays for highthroughput analyses of gene expression levels for more than a decade has resulted in the ability to generate a high volume of data on a number of species and tissue types.10-12 However, it is becoming more and more apparent that there is low correlation between mRNA levels and protein abundance,13,14 and, as a result, the development of tools for multiplex protein abundance determination is becoming increasingly important. While there is evidence that some degree of regulation of biological activity and cell differentiation is carried out at the level of gene transcription, most biological processes are carried out by, or are a subject of, the proteins that comprise the proteome of a living entity. Semiquantitative determination of gene expression levels via DNA arrays has been enabled by the basic underlying principle of DNA and RNA strand complementation. The high specificity of this interaction has been exploited for the design and implementation of high-throughput parallel analyses of thou* To whom all correspondence should be addressed. E-mail: gstchaga@ clontech.com.

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sands of genes. Today, scientists can accomplish this type of analysis with relative ease. While the implementation of stringent hybridization conditions for DNA analyses is practical due to the stability of the interacting strands to harsh conditions, the biological activity of most proteins is highly dependent on proteins’ secondary structure, that cannot withstand harsh storage and incubation conditions. Furthermore, while optimization and equalization of the hybridization properties (i.e., melting temperature, TM) of the immobilized capture oligos on DNA arrays is possible, the capture molecules that are used for determination of protein abundance (mainly antibodies) recognize their targets within a very broad range of binding affinities. In addition, there is no easy or universal way to amplify the signal correlating to the protein abundance in a high-throughput manner. Another major disadvantage of the currently available protein capture reagents is the lack of sufficiently high specificity and selectivity. While limited cross-reactivity of antibodies utilized in Western type analyses does not appear to be a major problem, such cross-reactivity can have a disastrous effect on the quality of data generated by their application in arrays. Our publication describes an experimental approach and mathematical algorithm that enables the use of hundreds, and even thousands, of existing capture reagents in highly parallel comparative analyses of protein abundance. Furthermore, we discuss some of the minimum requirements that should be met by capture reagents in order to ensure their inclusion for successful multiplex analyses of thousands of proteins. 10.1021/pr049776f CCC: $30.25

 2005 American Chemical Society

Relative Protein Abundance

2. Experimental Section 2.1. Methods. 2.1.1. Capture Reagents. All antibodies that were arrayed were monoclonal antibodies obtained from BD Biosciences Pharmingen, CA. The purified recombinant antigens used for screening and selection of array compatible antibodies were also provided by BD Biosciences Pharmingen, CA. All buffers for extraction, labeling, desalting, incubation, and washing were supplied commercially in the Ab Microarray 380 kit (Cat. No. K1847-1) from BD Biosciences Clontech, CA. Desalting PD-10 columns (Cat. No. 17-0851-01) and Monofunctional Cy3 (Cat. No. PA23001) and Cy5 dyes (Cat. No. PA25001) used for labeling of whole cell protein extracts and purified antigens were purchased from Amersham, NJ. 2.1.2. Western Analyses Equipment. Gel Running Apparatus: The Mini-PROTEAN 3 Electrophoresis Cell (165-3302) from BIO-RAD, CA, was used to run the 2-D/Prep gels. Gel Running Apparatus: The Criterion Cell (165-6001) from BIORAD, CA, was used to run the 26 well sodium dodecyl sulfate (SDS) electrophoresis. The transfer of the separated denatured protein bands to the Western membrane was performed with the Mini Trans-Blot Cell (170-3930) from BIO-RAD, CA. MultiScreen Apparatus for Western Blots: The Mini-PROTEAN II MULTI SCREEN (170-4017) from BIO-RAD, CA, was used for parallel Western analyses. 2.1.3. Cell Lines. HeLa cells from ATCC (Cat. No. CCL-2), Manassas, VA, USA were maintained in 150-mm culture plates at 37 °C in a humidified atmosphere of 5% CO2 in Dulbecco’s modified Eagle’s Medium (DMEM, 1 × Mod., w/ L-Glutamine, 4.5 g/L Glucose and Sodium Pyruvate) (234627) from BD (Becton, Dickinson and Company), NJ, supplemented with 10% v/v fetal bovine serum (FBS, 8630-1) from BD, NJ, and 1% v/v penicillin-streptomycin solution (P0781) from Sigma, St. Louis, MO. Jurkat cells, clone E6-1 (Cat. No. TIB-152) from ATCC Manassas, VA, were maintained at 37 °C in a humidified atmosphere of 5% CO2 in RPMI 1640 (1 × Mod., w/o LGlutamine) (234619) from BD, NJ, supplemented with 10% v/v fetal bovine serum (FBS, 8630-1) from BD, 1% v/v L-Glutamine, penicillin- streptomycin solution (G1146) from Sigma, St. Louis, MO. 2.1.4. Cell Pellets. Eight mL of 0.25% Trypsin-EDTA solution (T4049) at room temperature (RT) from Sigma, St. Louis, MO, was added to HeLa 150-mm culture plates, and incubated at RT for 10 min or until the cells were easily detached from the surface of the plate. Approximately 50-150 mg of cells was collected by centrifugation at 500 g and ambient temperature for 10 min in a preweighed centrifuge tube. Two combined 150mm HeLa culture plates or 50-60 mL of ∼1.5 × 106 Jurkat cells/ mL yield ∼150 mg of cells. The cell pellets were washed four times with 20 volumes of Dulbecco’s Phosphate Buffered Salt ×1 from BD, NJ, (Cat. No. 234626). The supernatant was discarded and the residual liquid was aspirated. Each tube was centrifuged for an additional 2 min, and any residual traces of liquid were aspirated. Each tube was re-weighed to determine the weight of the cell pellet. All cell pellets were frozen by placing them in -80 °C freezer. 2.1.5. Protein Extraction. 2.1.5.1. Cell Pellets. Cell culture test samples previously stored at -80 °C were moved on ice and 20 µL of extraction/labeling buffer from the Ab Microarray kit was added for each mg of cell pellet. The cell pellets were resuspended in extraction/labeling buffer by pipetting the

research articles buffer through the pipet tip several times until the samples were homogeneous. The suspensions were left to stand at RT for an additional 10 min then centrifuged at 10 000 × g for 30 min at 4 °C. The supernatants were placed in a clean microcentrifuge tubes at 4 °C. Protein concentrations were determined using the BCA (bicinchoninic acid) method. 2.1.5.2. Tissue Samples. Tissue samples of approximately 100-200 mg were placed in a pre-chilled mortar along with 0.25-0.50 g of alumina powder. The samples were ground to a paste after which 10 µL of extraction/labeling buffer was added for each mg of tissue. The buffer was mixed into the paste and then transferred to pre-chilled microcentrifuge tubes where an additional 10 µL/µg extraction/labeling buffer was added to the samples. The tubes were centrifuged at 10 000 × g for 30 min at 4 °C. The supernatants were placed in clean microcentrifuge tubes at 4 °C. Protein concentrations were determined using the BCA method. 2.1.6. Whole Cell Protein Labeling. Protein extracts were diluted with extraction/labeling buffer from the Ab Microarray kit to 1.1 mg protein/mL. Next, 110 µL of extraction/labeling buffer was added to one tube containing Monofunctional Cy3 dye. This solution was mixed quickly, but thoroughly, to suspend all of the dye in the tube. The tube was then spun for 10 s in a benchtop centrifuge. Fifty µL of that dye solution was aliquoted immediately into two separate 2-mL tubes. The same procedure was repeated with the Monofunctional Cy5 dye. Four-hundred fifty µL of the first protein extract was added to one tube containing Monofunctional Cy3 dye and one tube containing Monofunctional Cy5 dye, respectively. The same volume from the second protein extract was added to one tube containing Cy3 dye and one tube containing Cy5 dye, respectively. The labeling of the protein extracts was carried out for 1 h and 30 min at 4 °C in the dark. During this time, the tubes were inverted a few times every 20 min to mix the solution. Four µL of 1 M ethanolamine was added to each tube, and the solutions were incubated for 30 additional minutes. Again, the tubes were inverted a few times every 20 min to mix the solution. 2.1.7. Removal of Unincorporated Cy Dye. Four PD-10 columns were equilibrated with 10 mL of TST buffer (0.15 M NaCl, 10 mM Tris, 0.075% Tween 20) followed by 5 mL 1× of desalting buffer from the Ab Microarray kit (Cat No. K18471). Five hundred µL of labeled extract was applied to each column. 2.0 mL of 1× desalting buffer was applied to each column after the labeled sample was completely inside the column. The labeled high molecular weight components (including the proteins) were collected by elution with 2.0 mL of 1× desalting buffer. The collected eluates were placed on ice. 2.1.8. Protein Concentration and Substitution Degree of Labeled Protein Extracts. Protein concentrations of labeled samples were determined using the BCA (bicinchoninic acid) method. Sufficient BCA reagent mix was prepared at a 1:50 ratio of reagent A to B. BSA (Bovine Serum Albumin) standards were formulated in serial dilutions from 1.000 mg/mL to 0.0313 mg/ mL, and pipetted in duplicates of 25 µL into Corning 96-well flat bottom polystyrene plates. Samples were diluted 10:1, and pipetted in triplicates of 25 µL. 200 µL of BCA reagent mix was added to each standard/sample well. An additional blank for each labeled sample was used to eliminate the dye interference at 562 nm. Twenty-five µL of sample was added to 200 µL Ab Microarray desalting buffer. The plate was incubated for 30 min at 37 °C. After cooling to RT for 10 min, plates were read at Journal of Proteome Research • Vol. 4, No. 3, 2005 759

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Table 1. Model Protein Mix Experiment Components antigen

MW

DEMATIN nNOS/NOS Type1 ISGF3 p48 COL7A1 Pex1 SRP54 SH2-B MONA TRADD MYR6 Sum

39,400 46,300 64,750 64,920 65,740 65,540 54,800 54,740 59,020 38,210

Table 2. Model Protein Mix Experiment Setup

mix #1 pM

mix #2 pM

mix #3 pM

83.8 64.8 41.7 37.0 31.9 22.9 21.9 16.4 10.2 7.9 338.4

38.1 25.9 13.9 9.2 4.6 50.4 54.7 49.3 40.7 55.0 341.7

7.6 13.0 13.9 18.5 22.8 32.0 43.8 49.3 50.8 86.4 338.1

562 nm, and concentrations were determined by linear regression analysis. Labeled samples were diluted 5:1 in Ab Microarray desalting buffer. Optical density values for Cy3-labeled samples were read at 552 nm. Cy5-labeled samples were read at 650 nm. Dye-toprotein ratios were determined with the following calculation [Cy5 Dye] ) (A650)/250000 [protein extract] ) [(BCA value - (0.05‚(A650)]/60000* (Dye/Protein) ) [dye]/[protein extract] (Dye/Protein) ) (0.24‚(A650)/ [BCA value - (0.05‚(A650)] [Cy3 Dye] ) (A552)/150000 [protein extract] ) [(BCA value - (0.08‚(A552)]/60000* (Dye/Protein) ) [dye]/[protein extract]

Cy3 Mix

Cy5 Mix

#1 #2 #3

#1

#2

#3

0 X X

X 0 X

X X 0

Table 3. Protein Abundance Differences between Jurkat and HeLa Cells antigen name

normalized ratio INR (INR/INRmedian) WB

antigen MW, kD

higher abundance

TRAX DP-1 EGF Receptor Mre11 JNK1 DLP1 ZFP-37 VASP GS15 PMF-1 LEDGF SRPK1

0.12 0.23 0.29 0.39 0.40 0.41 0.45 0.49 0.49 0.51 0.52 0.53

6.48 3.20 2.55 1.92 1.88 1.82 1.67 1.54 1.53 1.48 1.44 1.42

+ + + + + + + + + + + +

33 52-55 180 81 46-55 84-79 67 46 15 23 52-75 92

Casein Kinase Ie PRK2 (PKN2/PAK-2) Tomosyn Mcl-1 eIF-5 MONA L-Caldesmon CD28 Flotillin 2/ESA MAP4

1.27 1.30 1.31 1.32 1.38 1.39 1.61 1.64 1.76 1.85

1.69 1.73 1.75 1.76 1.83 1.85 2.14 2.19 2.35 2.47

+ + + + NA + +

47 140 130 44 49 HeLa 38 80 44 42 200-220

Jurkat

(Dye/Protein) ) (0.40‚(A552)/[BCA value - (0.05‚(A552)] * is the assumed average molecular weight of the proteins in total extract 2.1.9. Internally Normalized Ratio. The Internally Normalized Ratio (INR) from any two samples (e.g., sample A and B) can be derived as follows: The ratio of the ratios from both slides can be defined as follows X/Y )

ACy5 BCy5 ACy5 ACy3 / ) * ) R1* R2 BCy3 ACy3 BCy3 BCy5

(1)

where X is the Cy5/Cy3 ratio obtained from slide #1, and Y is the Cy5/Cy3 ratio obtained from slide #2. In the ideal case, when the amount of any particular antigen is equal in both samples and the effect of differential labeling, solubility, affinity, etc. is the same for the antigen labeled with both labels (i.e., R1dR2), INR can be defined as follows INR ) xR * R ) xX/Y

(2)

2.1.10. Preparation of Model Antigen Mixes. Ten antigens were mixed according to the ratios presented in Table 1. Each mix was split into two equal portions and labeled as a mix with Cy3 and Cy5 dyes, respectively. The antigens were as follows: Dematin, nNOS/NOS Type1, ISGF3 p48, COL7A1, Pex1, SRP54, SH2-B, MONA, TRADD, and MYR6. In short, the mixes were dialyzed against the extraction/labeling buffer from the Ab Microarray kit and split into two equal 500 µg portions of total protein. A single tube of each NHS-Cy3 and NHS-Cy5 dye was used for the experiment. One hundred and 10 µL of extraction/ 760

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labeling buffer was added to each dye tube, the dye solution was mixed quickly, and 30 µL aliquot portions of each Cy dye was added to each mix aliquot in such a way that each mix was labeled with both labels. The labeling reaction was carried out for 1 h and 30 min at 4°C in the dark. The excess label was quenched by addition of 4 µL of 1M ethanolamine and subsequent incubation in the dark at 4 °C for an additional 30 min. The excess dye was removed by Size Exclusion Chromatography (SEC) against the desalting buffer from the Ab Microarray kit on PD-10 columns. 2.1.11. Model Antigen Mix Experiments. One experiment with nondiluted antigen mixes and two experiments with dilutions of 10 and 100 times the original mixes were carried out as follows: The nondiluted mixes were applied to the Ab Microarray slides at a total of 18 µg per channel. The first dilution resulted in an incubation of 1.8 µg, and the second dilution corresponded to an incubation of 180 ng of total protein per channel. This resulted in concentration ranges for the antigens between 600 pg/mL (for the 100 times dilution) to 660 ng/mL for the highest amount of antigen without dilution. The matrix in Table 2 was applied to the experiments with all three dilutions. After incubation of the samples for 30 min with the slides, the nonadsorbed material was removed by washing with the incubation buffer followed by seven (5 min each) washing steps with the washing buffers from the Ab Microarray kit The slides were dried by short 5 min centrifugation at 3000 × g and scanned on GenePix 4000B Axon scanner. 2.1.12. Validation of INR with the Same Cell Line. Cell extracts from HeLa and Jurkat cell lines prepared as described

Relative Protein Abundance

above were split into four equal portions, and two labeling reactions per dye were carried out with each cellular extract as described below. Each portion of HeLa extract was diluted to 1.1 mg/mL protein concentration. 0.5 mL of each portion was transferred to a new tube. One tube of each Cy dye was solubilized in 110 µL of extraction/labeling buffer. 50 µL of the solution was transferred quickly to two of the tubes containing the same cell extract. The same procedure was repeated with the second dye and the last two tubes of the same cell extract. The labeling reaction was carried out for 1 h and 30 min at 4 °C and then the residual reactive groups of the label was quenched with 1.0 M ethanolamine. The same overall procedure was repeated with the Jurkat extract. Two experiments were run by mixing the same HeLa cell extract labeled by both labels and 20 µg of total protein from the resulting mixes (or 10 µg of total protein per fluorescent channel) were incubated with two separate slides each. The incubation and washing were carried out as described above. The slides were dried by centrifugation and scanned on a GenePix 4000B Axon scanner. The same set of experiments was carried out with the Jurkat set of labeled cell extracts. A signal-to-background ratio higher than 2 was set as a threshold for choosing of the valid data points. 2.1.13. Determination of Relative Protein Abundance in HeLa and Jurkat Cells. HeLa and Jurkat cells were cultured and collected as described above. The total protein from approximately 100 mg of cell pellets collected from each cell line was extracted and labeled as described above with both Cy dyes. A total protein mix consisting of equal amounts of each (HeLa and Jurkat) of the protein extracts labeled with two different dyes (i.e., Cy5-HeLa and Cy3-Jurkat or Cy5-Jurkat and Cy3-HeLa) was prepared. A total of 20 µg protein from each mix (or 10 µg per cell extract) was incubated with one slide for 30 min. The nonadsorbed proteins were washed away according to the procedure described above, and the slides were dried and scanned on a GenePix 4000B Axon scanner. The resulting 16-bit gray scale Tiff files were analyzed either using the Axon GenePix 4.0 software or the Microarray software suite for IPLab (Scanalytics, Inc, VA). The resulting raw data reports were imported into the Microsoft Excel software package and the average intensity, background, and ratios from each antibody duplicate were used to determine the Internally Normalized Ratio (INR) from the two samples. A signal-to-background ratio higher than 2 was set as a threshold for selecting valid data points. However, if the signal/noise ratio was less than 2 for the same sample extract (i.e., Cy5-A and Cy3-A), but was above 2 for the other sample extract (i.e., Cy5-B and Cy3-B), the data points were used for further analyses. Once the INR values for all of the valid data points were determined, the antibodies were sorted by INR value. The median INR value was determined and appropriate brackets (i.e., 1.5 or 2 times higher/ lower than median INR) were applied to the data set. The collected data for all proteins that were found to be outliers by this method was evaluated for variability of the duplicates. 2.1.14. Western Validation. Criterion Precast Gel (345-0034) from BIO-RAD, CA, or Ready Gel (161-1138) from BIO-RAD, CA, was used for multi screen SDS electrophoresis. ImmunBlot PVDF Membrane for protein Blotting (0.2 mm) (162-0177) from BIO-RAD, CA, was used for transfer. See Blue Prestained Standard (LC5625) from Invitrogen, CA, was used as molecular weight standard. Primary detection monoclonal antibodies acquired from BD Biosciences Pharmingen, CA, were used at the recommended dilution specified on the Data Sheet of each

research articles particular Antibody. Affinity Purified Antibody Phosphatase Labeled Goat anti-Mouse IgG (H+L) from Kirkegaard & Perry Laboratories, Inc. (KPL), MD, (KPL Cat. No. 075-1806) was used as a secondary detection antibody with BCIP/NBT Phosphatase Substrate (50-81-07) from KPL. Protein Extracts were diluted with extraction/labeling buffer from the Ab Microarray kit (Cat No. K1847-1) to the same concentration. One volume of 5× SDS buffer (60 mM Tris-HCl (pH 6.8), 24% Glycerol, 2% SDS, 14.4 mM mercaptoethanol, 1% Bromophenol blue) was added to four volumes of each protein extract. The sample was boiled in a water bath for 5 min at 100 °C, cooled to ambient temperature, and then spun for 30 s on a benchtop centrifuge. The sample was either used immediately or stored at -20 °C. SDS electrophoresis was carried out with each type of gel according to the manufacturer’s instructions. 420 µL of sample was loaded on the Ready Gel, and 14 µL of sample per well was loaded on the Criterion Precast Gel. The protein bands from each gel were transferred to PVDF membranes according to the manufacturer’s instructions. After the transfer, the membranes were placed in blocking solution (2% w/v nonfat dry milk in TST) on a rocker platform at 4 °C overnight. The volume was sufficient to prevent the membrane from drying. The membranes obtained from the SDS electrophoresis on the Ready Gel were loaded onto the Mini-PROTEAN II MULTI SCREEN apparatus by placing the first lane (MW standards) over the MW marker. 560 µL of diluted primary antibody in blocking solution was loaded in the corresponding lane (the optimal antibody concentration was used as referenced from each individual antibody data sheet provided by BD Biosciences Pharmingen, CA). The membranes were incubated with the primary antibody on a rocker plate for 1 h at ambient temperature. Each lane was washed 3 times for a minimum of 5 min with blocking solution. Each wash was discarded. The membranes were removed from the Mini-PROTEAN II MULTI SCREEN apparatus and washed 3 times for a minimum of 5 min each with blocking solution. The secondary antibody was diluted in blocking solution to a final concentration of 0.05 µg/ mL and then added to the membranes and incubated for 1 h at ambient temperature on a rocker plate. The membranes were washed 3 times for 5 min in TST. Each wash was discarded. Substrate was added to each membrane and the membranes were developed on a rocker plate for 10-15 min at ambient temperature or until a strong background started to appear. The reaction was stopped with deionized water, and the membranes were air-dried. The membranes obtained from the SDS electrophoresis runs on the Criterion Precast Gels were cut to contain 3 lanes each: MW marker, HeLa, and Jurkat. Then each membrane was placed in 1.5 mL of their respective diluted antibody in blocking solution (the optimal antibody concentration was used as referenced from each individual antibody data sheet provided by BD Biosciences Pharmingen, CA). The membranes were incubated with the primary antibody on a rocker plate for 1 h at ambient temperature. Each lane was washed 3 times for 5 min with blocking solution. Each wash was discarded. The secondary antibody was diluted in blocking solution at a final concentration of 0.05 µg/mL and then added to the membranes and incubated for 1 h at ambient temperature on a rocker plate. The membranes were washed 3 times for 5 min each in TST. Each wash was discarded. Substrate was added to each membrane and the membranes were developed on a rocker plate for 10-15 min at ambient temperature or until a strong Journal of Proteome Research • Vol. 4, No. 3, 2005 761

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Figure 2. Validation of INR with the same extract from HeLa cells. The same HeLa cell extract labeled with both dyes was incubated on two separate slides (10 µg of total protein per fluorescent channel). The slides were scanned on a GenePix 4000B Axon scanner. A signal-to-background ratio higher than 2 was set as a threshold for choosing of the valid data points. The Cy5 to Cy3 ratio of the valid data points was plotted in Figure 2a for each slide and the obtained INR from the two slides was plotted in Figure 2b.

Figure 1. Validation of INR by model antigen mixes. Ten antigens in different relative amounts to each other (Table 1) were mixed in three different variations and labeled with Cy3 and Cy5 dyes. Each mix was run against a second mix in dual color, reverse color mode on two antibody array slides and the INR from each experiment was determined and plotted. To determine the dynamic range for quantitative determination of protein abundance each experiment was repeated using 10 and 100 time dilutions of the antigen mixes. The results were plotted side by side with those obtained from the starting mix concentrations.

background started to appear. The reaction was stopped with deionized water, and the membranes were air-dried.

3. Results 3.1 Labeling of Whole Cell Extracts. Multiple protein sources (serum, tissue culture extracts, tissues) were labeled with Cy3 and Cy5 reagents from different lots as described in the materials and methods section. The reproducibility of the labeling was followed by determination of the substitution degree and spectroscopic analyses of the labeled extracts. Not surprisingly, we observed significant variability of the labeling efficiency from dye to dye, as well as from different lots of the same dye (data not shown). This prompted us to look at alternative ways to ensure the lowest possible effect of the labeling step on the reproducibility of the array experiments. As a solution, we devised a dual color/reversed color array experiment on pairs of arrays. 762

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3.2. Model Antigen Mix Experiments. To validate the relevance of the proposed experimental setup we devised a series of experiments that utilized model antigen mixes containing varying amounts of the antigens. Each model mix was labeled with both fluorescent dyes. It is important to point out that the labeling reactions were carried out on the whole antigen mixes rather than on the individual antigens in order to mimic conditions that are present during labeling of whole cell extracts as closely as possible. After carrying out the preparation of the model antigen mixes and their dual color labeling, the mixes were run against each other (see Table 1 for the total theoretical amount of each antigen per channel for the experiment without dilution) as described in Table 2. A total of six slides containing all 384 antibodies printed in duplicates were incubated per dilution, and the raw data obtained from each pair of mixes was used to calculate the INR (experimental) value for each antigen. These values were compared to the theoretical values for the ratio of each antigen in every pair of model antigen mixes. The comparison of the theoretical and experimental values is presented in Figure 1. There is a good correlation of the theoretical values and the experimental INR values for all pairs of antigen mixes with the exception of that observed for MYR6 in the pairs of Mix 1 versus Mix 2, and Mix 3 versus Mix 1. Despite the significant difference between the theoretical and experimental values, even these two data points indicate relevant differences in the pairs, pointing correctly that MYR6 is lower in abundance in Mix 1 as compared to Mix 2, and higher in abundance in Mix 3 as compared to Mix 1. Interestingly enough, the INR value of the ratio of MYR6 in Mix 3 versus Mix 2 is very close to that of the

Relative Protein Abundance

Figure 3. Validation of INR with the same extract from Jurkat cells. The same Jurkat cell extract labeled with both dyes was incubated on two separate slides (10 µg of total protein per fluorescent channel). The slides were scanned on a GenePix 4000B Axon scanner. A signal-to-background ratio higher than 2 was set as a threshold for choosing of the valid data points. The Cy5 to Cy3 ratio of the valid data points was plotted in Figure 2a for each slide and the obtained INR from the two slides was plotted in Figure 2b.

theoretical ratio. One could speculate that a slight crossreactivity of the antigen with other antibodies on the array and/ or differential yield during labeling and desalting of MYR6 when present in low abundance in the mix is the cause of not very strong quantitative correlation. Another antigen that apparently does not give a good quantitative estimate of its relative abundance without dilution

research articles is TRADD. It appears, however, that at 10 times dilution the INR value for TRADD is very close to that of the expected theoretical ratio. A possible explanation is that at the high concentration levels (Mixes 2 and 3) the capture antibody on the array is saturated and this results in lower dynamic range in detection. However, the INR value points again to the correct higher relative abundance of TRADD in Mixes 2 and 3 as compared with Mix 1. In general, with the exception of the results for TRADD, the predictive quantitative value of all other antigens decreases with the dilution of the antigens. Despite that, even at hundred times dilution of the original mixes, there is a good correlation between the experimental (INR) and theoretical values. This represents a fairly broad dynamic range in which the protein sample can be analyzed. To ensure a broader range, one could run serial dilutions of the analyte in a separate parallel experiment. 3.3. Validation of INR with the Same Cell Line. A number of publications discussing the relevance of detected differential gene expression point out that the percentage of valid changes varies between 3 and 5%. However, utilizing a single color system or a dual color system on single array results in the potential identification of significantly higher number of potential targets. To minimize the generation of false positive results, we looked at the effect of the INR experimental setup on experimental data generated from the same cellular extract. The result of the experiments on validating the effect of the experimental setup and INR algorithm on the quality of generated data is presented in Figures 2 and 3. Figures 2a and 3a present the variability in determining protein abundance if the experiments are carried out on a single slide as dual color experiments. There are a number of false positive ratios that point out apparent changes in the abundance of proteins in

Figure 4. Application of INR experimental setup to determination of relative protein abundance in HeLa and Jurkat cells. HeLa and Jurkat cell extract labeled with both labels were mixed and incubated at 10 µg of total protein per fluorescent channel on two separate slides (slide 1 with HeLa-Cy5 and Jurkat-Cy3 mix, slide 2 with HeLa-Cy3 and Jurkat-Cy5 mix respectively). The slides were processed as described in Methods and scanned on a GenePix 4000B Axon scanner. The pseudo-color representation of the 16-bit Tiff files acquired from both slides depicts the relative protein abundances in HeLa versus Jurkat cells. Red arrows point to antibodies detecting proteins with higher apparent abundance in the Cy5 channel, while green arrows point to antibodies detecting proteins with higher apparent abundance in the Cy3 channel. Yellow arrows point to antibodies detecting proteins with no apparent difference in the abundance between the two cell lines. Journal of Proteome Research • Vol. 4, No. 3, 2005 763

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Figure 5. Validation of the antibody array INR data by western analyses. A number of differences in the protein abundance detected by the antibody array experiment were validated by western analyses. The name of the antigen analyzed is under each pair of lanes, H and J above the pairs of lanes denote HeLa and Jurkat extracts, respectively. The arrows point to the band with expected molecular weight except in two cases (PRK2 and MONA) where non specific bands can contribute to false positive or negative signals if these non specific polypeptides are detected in their native form on the antibody array as well.

the same cellular extract during the labeling, desalting, and incubation with the Ab Microarray slide. However, when the INR algorithm is applied (Figures 2b and 3b), one can observe a significant decrease in false positive data points. In addition, all of the valid data points (S/N g 2) are clustered between values of 0.8 and 1.2. These results increase the confidence level of predicting valid differences in abundance for INR data points that lay as close as 0.7 and 1.3. A number of similar experiments were also carried out with tissue extracts and were found to support the results obtained with cell extracts (results not shown). It is important to point out that this result is reproducible with any type of sample that has been labeled properly and yields protein recovery greater than 85% of the initial protein amount (before labeling) after desalting. 3.4. Determination of Relative Protein Abundance in HeLa and Jurkat Cells. The application of the INR principle to the determination of relative protein abundance levels in different samples is exemplified in Figure 4. As one can observe, there 764

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are a number of differences in the abundance of proteins in Jurkat and HeLa cells. Some of these changes are apparent even to the naked eye when comparing the switch in color from red to green and the reverse in the pseudo color presentation of the signal ratios between slide one (Cy5-Jurkat vs Cy3-HeLa) and two (Cy5-HeLa vs Cy3-Jurkat). However, there could be valid abundance differences that are not apparent by visual observation. While particular antibody-antigen containing spots do not reverse color from the first to the second slide, the use of INR can point to a valid abundance difference between the two samples. Results such as these, when obtained from a single slide dual color experiment could actually indicate a difference in protein abundance that is diametrically opposite to the real one. Table 1 presents the INR values as well as the normalized ratios based on the value obtained from dividing or multiplying the INR for these proteins by the median INR for the whole slide. The validity of the observed abundance changes in the array experiment was qualified by Western blot analyses of the

Relative Protein Abundance

protein levels in the cellular extracts from Jurkat and HeLa. The results of these analyses are presented in the WB column in Table 1 and Figure 5. The Western results confirm the array data in the majority of cases: seventeen observed changes are confirmed by Western, one antibody did not have sufficient sensitivity in Western to detect the target antigen, and three Western results show opposite ratios of the antigen to that observed in the array. The data from MONA is somewhat difficult to interpret. A band with the expected MW of 38 kD has median intensity in the Jurkat sample, but in HeLa there is a strong band with MW at around 30 kD, which might be a proteolysis product. A set of different cell extracts from Jurkat and HeLa that were analyzed for the presence of MONA by Western blot seems to suggest that the lower band might be a proteolysis product because it is present in both cell extracts (data not shown). The situation is exacerbated by the presence of three dominant bands in both samples that have MWs higher than the expected 38 kD. The same is true for PRK2, where a strong band at 140 kD is detected in Jurkat, and a stronger band at 36 kD is detected in HeLa. There is 80% correlation between the antibody array data and the data obtained from Western analyses if the Western result from MONA is interpreted as not supporting the array finding.

4. Conclusions This manuscript presents an experimental layout and algorithm for the determination of the relative abundance of hundreds, and even thousands, of proteins in two samples. The experimental setup ensures that there is low, or no, false positive targets and enables the use of hundreds of antibodies in array format for multi parallel analyses. There has been significant progress in multiplexing immunoassays utilizing a parallel ELISA format, and a number of studies have been published in this regard. However, given the complexity of any biological sample and the inherent crossreactivity of the capture and detection reagents, all applications so far have been restricted to at most 40 analytes per assay compartment. While multiplex and multi parallel ELISA assays are important enabling tools, obtaining a screen shot of the proteome of a given cell type, tissue, or biological fluid is not the goal of these assays. Increasing the number of protein analytes to hundreds, and even thousands, is difficult because of the crossreactivity problems, detection restrictions, and the need to utilize universal conditions for all proteins that have to be analyzed. Given the versatility of the properties of the proteins, the last condition is limiting in any system that uses native proteins. An added difficulty is the need to deposit the capture reagents on a solid surface and protect their biological activity in a reproducible manner to allow data comparison between different experiments. To address some of these challenges, we have introduced the use of the Internally Normalized Ratio experimental setup and analyses algorithm and have shown that it significantly reduces the number of false positive targets. Its application enables the use of hundreds, and even thousands, of capture reagents as well as the multi parallel analyses of the relative abundance of their corresponding antigens.15-20 We have already expanded the array to include more than 500 antibodies (data not shown), and it appears that there is little, or no, technology restriction on continuing to scale-up the number of analytes. We believe that the ideas described here will enable the development of even larger arrays for scientific discovery

research articles

Figure 6. Schematic presentation of the complexity of multiplex immunoassays.

experiments. Some important aspects of the type of assay that we discuss are as follows: 4.1. Strengths and Weaknesses of Multi Parallel Analyses. We wish to introduce a somewhat modified process parameter pyramid (Figure 6). There are number of significant factors that are not reflected here but these five are sufficient to exemplify the complexity in the development of multi parallel assays. As one chooses the point at which the assay has to operate, one inevitably makes compromises along all eight axes of the pyramid. It is obvious that achieving a high number of parallel analyses leads to a compromise with one of the four other parameters: quantitation, sensitivity, dynamic range, and speed. In reality, we have no control over the sample complexity if our goal is to carry out global analyses of the proteome. The speed of the immunoassay is usually inversely proportional to the sensitivity, but a sensible compromise can be made. The sensitivity is pre-defined for each capture reagent and cannot be modified significantly. A global amplification scheme that can be applied to the whole set of analytes is possible and practical with single color detection. Running a single color detection rather than a competitive dual color assay for relative abundance presents challengesstechnical problems, such as unequal capture deposition on different arrays and differential cross-reactivity, affect the quality of the data strongly. We believe that, for the type of analyses for which the Ab Array is designed, the best choice is to utilize dual color/reverse color experiments (Figure 2). Multiplex competitive analyses of directly labeled protein samples present the following challenges: sReproducible labeling: This is addressed by dual color/ reverse color experimental design that compensates for variation in labeling by using both labels on both samples and the same labeling solution for both samples. sReproducible removal of excess label: This is addressed by dual color/reverse color experimental design by analyzing labeled samples with comparable losses for each protein solution labeled with the same dye. sLabel interference effects with epitope presentation: This is addressed by control over the level of label incorporation by optimization of the amount of the incorporated dye in both samples (not shown) and by utilizing samples with very similar incorporation levels of the same type dye. 4.2. Contradictory Data between Western and Array Analysis. There are some cases when the Western analysis indicates diametrically opposite results compared to the array assay. Possible scenarios that might lead to this include: 1. There is a differential extraction efficiency and solubility of the antigen under native and denaturing conditions: If this Journal of Proteome Research • Vol. 4, No. 3, 2005 765

research articles is the case, both assay systems indicate the real abundance differences in the respective extracts; however, they are applied to two different extracts. It should be noted that during the development of the extraction procedure for the antibody arrays, we observed that a protein, such as cytochrome P450, was extracted more efficiently by the array extraction buffer than by the SDS boiling step used to prepare samples for SDSelectrophoresis (data not shown). The real abundance of the analyte in the original sample has to be determined by followup experiments. 2. The antibody used in the array could recognize a 3D epitope better than a linear peptide: In this case, the antibody array data should point to the real abundance difference between the two samples. The Western analysis might point to the opposite result (e.g., if a linear peptide that mimics the 3D structure becomes available after denaturation and has similar size as the antigen). An example of this might be the lack of sensitivity to detect the antigen in Western assay (the case of CD28). 3. The contribution of cross-reactivity to total amount of the signal on the antibody-antigen spot is higher than 50%: While the specific antigen in one of the samples has decreased, the amount of cross-reacting antigens in the same sample has increased, exceeding the rate of that of the decrease of the antigen. In this case, the array data would point to relative abundance difference that is opposite to the real one. It could be difficult to determine if a native or denatured assay is better for a given analyte. Development of an ELISA test for this particular antigen might be helpful. Immunoprecipitation and follow up analyses by ELISA and Western might help to determine the validity of the data. 4.3. Single Capture vs Antibody Pair Detection. There is a prevailing opinion that the use of pairs of capture and detection antibodies increases the quality of the data as compared to the use of a single capture antibody 21-23. The two types of assays offer different advantages and disadvantages in regard to performance under specified assay conditions: The crossreactivity from one of the samples is significantly higher than that from the second sample. This is a rare case when related samples are applied to the array, but can have an effect on the quality of data obtainable from a single capture molecule as compared to an antibody pair detection. If the nonspecific binding occurs at the Fc region of the capture antibody, the antibody pair detection approach offers an advantage since it will not detect, or be influenced by, the cross-reacting protein(s). However, if the cross-reactor is one of the other target analytes in the mix, a nonspecific signal will be generated by the primary detection antibody for this cross-reacting antigen. A signal like that cannot be differentiated in a single color experiment and can contribute to higher than the real abundance being detected. Such problems can be minimized with both types of assays by printing high density spots where the statistical representation of exposed Fc regions is less than 30% or simply by utilizing Fab fragments as capture reagents. In pair detection, additional factors, such as capture antibody amount variability between different arrays, sample amount variability, primary and secondary detection antibody amount variability and cross-reactivity of the detection pairs can influence the quality of the assay. Relative protein abundance determination from a competition of the antigen from both samples is also possible with a single capture antibody and dual color setup if the cross-reactivity signal contributes to less than 50% of the detected signal. 766

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Andersson et al.

If the nonspecific binding occurs at or close to the CDR regions, it cannot be compensated by pair detection. However, relative protein abundance determination from a competition of the antigen from both samples is still possible with a single capture antibody and dual color setup if the cross-reactivity signal is less than 50% of the detected signal. In a recent paper in Science, James et al. discuss the conformational diversity of some antibodies and the effect this diversity has on antibody multispecificity 24. While the broadly accepted idea of “induced fit” could be applied to a part of the binding process between antibody and antigen, it is just a part of the process. As James et al. demonstrate, there is an equilibrium between distinct conformations of the antibody molecule, and this equilibrium can be influenced by the binding of an antigen to its specific conformation (i.e., by depletion of the antibody of the isotype involved in the antigen binding, more molecules of the second isotype are assuming the conformation that binds the antigen). This effect has the following implications: A. Effective concentration differences between the antigen and cross-reacting component(s) play a significant role in the quality of array data. B. In solution (homogeneous), the effect of binding between antibody and antigen might be very different from that of immobilized antibody and antigen in solution. Due to the high density of antibodies in array spot, the cooperative effect of the antigen binding to the proper conformation of one antibody molecule might induce a significant amount of surrounding antibodies to shift to the same conformation. This might explain our observation of very high sensitivities of some antibodies on the array toward their respective purified antigensssometimes significantly higher than corresponding sensitivity in Western analyses. In any case, while the single capture assay would not provide quantitative data, it could indicate which sample has the antigen in higher abundance. There is no doubt of the value of tools that enable us to look at hundreds, and even thousands, of protein analytes at the same time. It is commonly accepted that no single analyte can have absolute predictive value on important biological questions such as cell death, proliferation, aging, cancerogenesis, communication, etc. Given the heterogeneity of higher forms of life, development of accurate assays for these and other biological processes is even more challenging. Multi parallel analyses seem to hold the promise to enable us to decrease both the costs and the time for analyses. We have already seen the first attempts for the application of DNA arrays as predictive and diagnostic tools. It is desirable to develop multi parallel analyses for the machinery and the major building blocks of living mattersnamely proteins. However, the enormous complexity and versatility of proteins is a major stumbling block in the development of such assays. Extremely high dynamic ranges of their concentrations from various sources (1012 for plasma and serum) further hinder the development of such assays. Nevertheless, the use of 2D gel electrophoresis and LC/ MS/MS have helped significantly in our understanding of the composition of the protein samples at least for high and medium abundance proteins within a broad range of isoelectric points. The use of antibody arrays has a complementary value in enabling us to look at low abundance proteins, membrane proteins, and proteins with extreme pI values. In addition, their application as primary capture arrays for native proteins enables further studies of protein-protein interactions. Their application for determination of post-translational modifica-

research articles

Relative Protein Abundance

tions of their respective antigens is another extremely important goal, and our group is working very hard in trying to develop these types of assays. Abbreviations: BD, Becton, Dickinson and Company; INR, Internally Normalized Ratio; MW, Molecular Weight; PBS, Phosphate Buffered Saline; RT, Room Temperature; SEC, Size Exclusion Chromatography; SDS, Sodium Dodecyl Sulfate; Tris, Tris(hydroxymethyl)aminomethane; TST, 10 mM Tris, 0.15 M NaCl, 0.075% Tween 20.

Supporting Information Available: HeLa-Cy5 vs Jurkat-Cy3 Raw (1 table). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Haab, B. B. Advances in protein microarray technology for protein expression and interaction profiling. Curr. Opin. Drug Discov. Devel. 2001, 4, 116-123. (2) Arenkov, P. et al. Protein Microchips: Use for Immunoassay and Enzymatic Reactions. Anal. Biochem. 2000, 278, 123-131. (3) Bussow, K.; Konthur, Z.; Lueking, A.; Lehrach, H.; Walter, G. Protein array technology. Potential use in medical diagnostics. Am. J. Pharmacogenomics 2001, 1, 37-43. (4) Huang, R. P.; Huang, R.; Fan, Y.; Lin, Y. Simultaneous detection of multiple cytokines from conditioned media and patient’s sera by an antibody-based protein array system. Anal. Biochem. 2001, 294, 55-62. (5) Lueking, A.; H. M.; Eickhoff, H.; Bussow, K.; Lehrach, H.; Walter, G. Protein microarrays for gene expression and antibody screening. Anal. Biochem. 1999, 270, 103-111. (6) Robinson, W. H.; Steinman, L.; Utz, P. J. Protein and peptide array analysis of autoimmune disease. Biotech. Suppl. 2002, 66-69. (7) 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. (8) Bock, C. et al. Photoaptamer arrays applied to multiplexed proteomic analysis. Proteomics 2004, 4, 609-618. (9) Shao, W. et al. Optimization of Rolling-Circle Amplified Protein Microarrays for Multiplexed Protein Profiling. J. Biomed. Biotechnol. 2003, 2003, 299-307.

(10) Ramsay, G. DNA chips: state-of-the art. Nat. Biotechnol. 16, 4044 1998. (11) Hughes, T. R.; Shoemaker, D. D. DNA microarrays for expression profiling. Curr. Opin. Chem. Biol. 2001, 5, 21-25. (12) Chittur, S. V. DNA microarrays: tools for the 21st Century. Comb. Chem. High Throughput Screen 2004, 7, 531-537. (13) Aebersold, R.; Rist, B.; Gygi, S. P. Quantitative proteome analysis: methods and applications. Ann. N. Y. Acad. Sci. 2000, 919, 33-47. (14) Chen, G. et al. Discordant protein and mRNA expression in lung adenocarcinomas. Mol. Cell Proteomics 2002, 1, 304-313. (15) Anderson, K.; Potter, A.; Baban, D.; Davies, K. E. Protein expression changes in spinal muscular atrophy revealed with a novel antibody array technology. Brain 2003, 126, 2052-2064. (16) Marienfeld, C. et al. Translational regulation of XIAP expression and cell survival during hypoxia in human cholangiocarcinoma. Gastroenterology 2004, 127, 1787-1797. (17) Yamagiwa, Y.; Marienfeld, C.; Meng, F.; Holcik, M.; Patel, T. Translational regulation of x-linked inhibitor of apoptosis protein by interleukin-6: a novel mechanism of tumor cell survival. Cancer Res. 2004, 64, 1293-1298. (18) Gosmanov, A. R.; Umpierrez, G. E.; Karabell, A. H.; Cuervo, R.; Thomason, D. B. Impaired expression and insulin-stimulated phosphorylation of Akt-2 in muscle of obese patients with atypical diabetes. Am. J. Physiol. Endocrinol. Metab. 2004, 287, E8-E15. (19) Hudelist, G. et al. Use of high-throughput protein array for profiling of differentially expressed proteins in normal and malignant breast tissue. Breast Cancer Res. Treat. 2004, 86, 281291. (20) Ghobrial, I. M. et al. Proteomic analysis of mantle cell lymphoma by protein microarray. Blood 2005. (21) Schweitzer, B. et al. Multiplexed protein profiling on microarrays by rolling-circle amplification. Nat Biotechnol. 2002, 20, 359365. (22) Nielsen, U. B.; Geierstanger, B. H. Multiplexed sandwich assays in microarray format. J. Immunol. Methods 2004, 290, 107-120. (23) Turtinen, L. W.; Prall, D. N.; Bremer, L. A.; Nauss, R. E.; Hartsel, S. C. Antibody array-generated profiles of cytokine release from THP-1 leukemic monocytes exposed to different amphotericin B formulations. Antimicrob. Agents Chemother. 2004, 48, 396403. (24) James, L. C.; Roversi, P.; Tawfik, D. S. Antibody multispecificity mediated by conformational diversity. Sci. 2003, 299, 1362-1367.

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