Patent Application
20120220489
Reverse phase protein arrays (RPA) have been developed and established in the recent years as a convenient method to analyze focused sets of proteins representing key analytes of different signal transduction cascades in minute amounts of biological samples (e.g. cell lysates, tissue lysates, or body fluids). Relative differences of protein expression, representing not only the abundance of specific key proteins, but also activated, post-translationally modified (e.g. phosphorylated) forms of such key proteins can describe and classify e.g. specific treatment effects of pharmaceutical compounds given to cell cultures e.g. inhibitory effects of drug candidates on kinases, or describe and classify different disease states e.g. sub-types of tumors in their different progression states. RPA can perform comparative measurements of many samples in parallel, e.g. samples from differently treated cell cultures or samples from different disease populations. Significant changes of protein expression or protein activation patterns to be found in distinct sample cohorts will foster e.g. the identification of most efficient drug candidates, the elucidation of treatment induced mode-of-action schemes or the discovery of new diagnostic/prognostic disease markers.
Immunoaffinity assays such as used in Reverse Phase Protein Arrays (RPA) are based on specific interactions between an affinity reagent and a protein of interest. The assay comprises the immobilization of the biological samples on the array forming the sample spots. The sampled array is incubated with an affinity reagent, i.e. an antibody, and the subsequently formed complex of affinity reagent and protein of interest is measured by the generated detection signal e.g. a luminescence signal. Each array is stained with an analyte-specific affinity reagent, which can be labeled or is incubated with a secondary detection reagent. Formed complexes are detected by various means (colorimetric, fluorescence, chemiluminescence etc.). Typically RPA measure relative changes of expression or activation signals between different samples.
The quantitative analysis of samples requires the use of calibration reagents. Currently, for protein analytes, the calibration reagents are recombinant proteins having the same amino sequence as the analyte. For example, patent application WO2007/048436A1 describes calibration curves for Reverse Phase Protein Microarrays whereby different concentrations of purified protein of interest (Akt) were added to spotting buffer comprising BSA or rat serum. However, the production of recombinant protein presenting the correct epitope is time-consuming and often not successful. In particular for phosphorylated epitopes, so far no reliable calibration reagents are available.
Therefore, there is a need for a reagent designed to provide universal applicability with choosable specificity for the different analyte epitopes of interest. This would allow to calibrate results from experiments performed, e.g. at separated times, by different lab personal, on different devices or on arrays constructed in different print runs. Also the linear range of the protein-specific RPA signals to be generated by the respective affinity reagent can be optimally pre-defined.
Therefore, the present invention provides a calibration reagent comprising a peptide which is attached via a linker to a protein carrier, wherein said peptide comprises an epitope of interest. Preferably, said epitope of interest is phosphorylated.
With this calibration reagent reliable standard curves can be generated for quantifying protein with an RPA or another affinity assay. RPAs are constructed by the deposition of small sample volumes e.g. of cell or tissue lysates, onto highly binding substrate surfaces using often a robotic microarrayer. Each lysate spot on the substrate contains the full complement of cellular proteins and analytes. Hundreds of samples can be spotted in parallel into one microarray allowing high throughput cross-comparisons of samples in the same assay. Replicate arrays containing the same set of samples, can be easily produced from the same initial volume of sample material, since consumption of sample volume per spot is extremely low.
The calibration reagent of the current invention is particular useful for quantifying proteins which comprise a phosphorylated epitope of interest.
The term “epitope of interest” refers to a part of a polypeptide which is recognized by the affinity reagent of interest. The affinity reagent of interest is preferably specific for the epitope of interest.
The term “epitope peptide” as used herein refers to the peptide comprising the epitope of interest. The epitope peptide is preferably between 12 and 25 amino acids long. More preferably, the length of the peptide is 12 to 20, most preferably 14 to 17 amino acids long.
The epitope of interest can be modified, e.g. phosphorylated. The term “phosphorylated epitope” as used herein refers to an epitope which comprises at least one amino acid with a phosphate group. Preferably, the epitope of interest comprises 1 to 5 phosphorylated amino acids. Preferably, the position of the modified amino acid is approximately in the middle of the epitope peptide. Fore example in a peptide of 15 amino acids length, the modified amino acid is preferably at position 7, 8 and/or 9 (see
The epitope peptide is covalently bound to the protein carrier via a linker (see
The epitope peptide can be attached to the protein carrier in essentially two steps:
Step 1) The linker is conjugated to the epitope peptide, wherein said linker is preferably labeled with a tag. The linker can be attached to the N- or C-terminus of the peptide. Preferably, the linker is attached to the N-terminus of the peptide.
Step 2) the free end of the linker is conjugated to the protein carrier.
The linker or spacer is a peptide comprising 2 to 10, preferably 2 to 5, more preferably 3 to 4 natural or unnatural amino acids. Natural amino acids are naturally occurring amino acids such as in particular alanine, cysteine, lysine, histidine, arginine, aspartate, glutamate, serine, threonine, methionine, glycine, valine, leucine, isoleucine, asparagine, glutamine, proline, tryptophane, phenylalanine, tyrosine. Unnatural amino acids are amino acids which do not naturally occur. Examples for unnatural amino acids are 8-amino-3, 6 dioxa-octanoic acid (Doa) and aminooxy-acetic acid.
The linker is hydrophilic and can comprise only natural amino acids or only unnatural amino acids or a mixture of both, natural and unnatural amino acids. Preferably, the linker comprises one or more of the following natural amino acids: cysteine, lysine, histidine, arginine, aspartate, glutamate. Also preferably, the linker comprises one or more Doa. More preferably, the linker is Cysteine-Doa-Doa.
Preferably, the linker is labeled with Dabsyl.
Methods to produce peptides with a specific amino acid sequence are well known to the skilled person in the art. A suitable method is e.g. is the solid phase synthesis described in Merrifield, Science 1986, 232:341-347 (method) and Carpino et al., J. Am. Chem. 1990, 112: 9651-52 (reagents).
The protein carrier is a protein which unspecifically binds to surfaces. Preferably, the protein carrier is a protein of at least 20 kDa and shows no or low cross reactivity with the affinity reagent used in an affinity assay. The protein carrier is preferably an albumin, more preferably a serum albumin, such as e.g. bovine serum albumin (BSA) or human serum albumin. The preferred serum albumin is BSA.
An “affinity reagent of interest” is a reagent which recognizes and binds the epitope of interest. Preferably, the affinity reagent of interest is specific and selective for the epitope of interest. The affinity reagent can be an antibody, an aptamer, or a designed ankyrin repeat protein (DARPin). Preferably, the affinity reagent is an antibody.
An “antibody of interest” can be any antibody. Preferably, said antibody is an IgG antibody, more preferably a monoclonal antibody. The antibody of interest includes but is not limited to humanized antibody and rodent antibody. A rodent antibody includes but is not limited to a mouse, rabbit and rat antibody. Preferably, the antibody is a rabbit monoclonal antibody.
An “aptamer of interest” is a single-stranded RNA or DNA oligonucleotide 15 to 60 base in length that bind with high affinity to the epitope of interest.
A “designed ankyrin repeat protein” or “DARPin” is a binding molecule comprising at least one ankyrin repeat. An ankyrin repeat is a motif in proteins consisting of two alpha helices separated by loops, which can be selected to recognize specifically a wide variety of target proteins. The typical length of an ankyrin repeat is 33 amino acids. Unlike antibodies they do not contain any disulfide bonds and are found in all cellular compartments.
Furthermore, the present invention provides the use of the calibration reagent as described above for the generation of a standard curve. The present invention provides a method for generating a standard curve comprising the steps of:
a) immobilizing the above described calibration reagent in two or more concentrations on an array,
b) incubating said array with a detectable affinity reagent of interest,
c) measuring the signal intensity of the bound affinity reagent for each of the two or more concentrations of the calibration reagent, and
d) correlating the signal intensity with amount of epitope of interest.
A standard or calibration curve is a quantitative research tool, a method of plotting assay data that is used to determine the concentration of a substance, i.e. the concentration of the epitope of interest.
The term “bound affinity reagent” refers to the affinity reagent forming a complex with a protein or peptide comprising the epitope of interest. Formed complexes are detected by various means such as for example colorimetric, fluorescence, or chemiluminescence.
An affinity reagent can be detected, by a detectable label attached to the affinity reagent. Preferably, said label is a fluorophore, allowing thereby to determining the amount of bound antibody by the fluorescence intensity. Other suitable labels are e.g. alkaline phosphatase (AP) and horseradish peroxidase (HRP).
An affinity reagent can also be detected by a secondary detection reagent. A secondary detection reagent is a labeled molecule which selectively binds the affinity reagent. The bound affinity reagent on the microarray can be detected for example by using a second antibody or a Fab fragment, which is labeled and recognizes species-specific epitopes of the affinity reagent. Suitable labels include but are not limited to fluorophore, biotin, horseradish peroxidase, and isotopes. Preferably, the secondary detection reagent (e.g. a secondary antibody) is labeled with a fluorophore.
The amount of affinity reagent bound to the calibration reagent is preferably detected by an optical signal such as a fluorescence signal.
The amount of bound affinity reagent of interest is correlated with amount of epitope of interest by measuring the detectable signal of the affinity reagent and attribute each signal a concentration of the epitope of interest. These results are displayed in a standard curve. A standard curve can be drawn by plotting the determined amount of bound affinity reagent of interest for each concentration of the epitope of interest (on the Y axis) versus the concentration of the epitope of interest (on the X axis). The amount of bound affinity reagent is usually displayed as the strength of the detected signal (signal intensity). Preferably said signal is an optical signal, more preferably fluorescence intensity. Typically, for the purpose of generating a standard curve, the spots on the array comprise different concentrations of the calibration reagent, preferably as a serial dilution (e.g. a series of two-fold dilution).
The concentration of the epitope of interest in a known concentration of calibration reagent is obtained by determining the peptide:carrier ratio, which is the number of peptides conjugated to one protein carrier.
Methods for determining the peptide:carrier ratio are well known to the skilled person in the art. A suitable method is e.g. a method comprising the following steps: step 1: determining the conjugated peptide concentration by for example photometric absorbance measurement, whereby the peptides or the linker attached to the peptides are preferably labeled with a tag (e.g. Dabsyl); step 2: determining the total protein concentration of the conjugated product via Bradford test and step 3: calculating the peptide:protein ratio. Preferably, the linker is labeled with tag such as e.g. Dabsyl, which allows to determine the peptide:protein carrier ratio. Suitable ratios for use in the methods of the invention can be up to 10 and higher. Preferably, the ratio is lower than 3, more preferably, the ratio is equal to or lower than 1, most preferably the ratio is between 0.3 and 1.
The affinity reagent of interest is incubated on the array for at least 30 minutes, preferably for more than 1 hour, more preferably for 1 to 16 hours, most preferably about 12 hours (12 hours ±30 minutes). The excess of affinity reagent is removed and preferably the array is washed before measuring the signal intensity.
An array is a solid support with has a hydrophobic surface, allowing the binding of proteins to the surface. Arrays for RPAs and other affinity assays are commercially available and well known to the skilled person in the art. The calibration reagent is immobilized on the array by interaction of the carrier protein with the surface of the array. To avoid unspecific binding to the hydrophobic surface the spotted array preferably is subsequently coated with an unspecific protein, such as e.g. BSA.
The calibration reagent is applied on the array in two or more concentrations. Preferably, the applied concentrations form a dilution series (e.g. a dilution series of 1:2, 1:5, or 1:10). The calibration reagent is preferably applied in at least three different concentrations. More preferably, the calibration reagent is applied in 3 to 20 different concentrations, even more preferred are 5 to 15 different concentrations.
The calibration reagent can be applied at the desired position on the array as a spot. The calibration reagent is typically solved in a buffer. A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. A suitable buffer is e.g. CSBL spotting buffer (Product number 9020, Zeptosens, Witterswil, Switzerland). In a preferred embodiment said buffer comprises matrix proteins. A preferred matrix protein is BSA, more preferably the matrix protein is acetylated BSA. Typically, the applied calibration reagent solution is allowed to dry before incubating the array with the affinity reagent of interest.
The spots of the calibration reagent on an array are typically arranged in fields. Array fields can form geometrical areas such as e.g. squares, rectangles, circles, and triangles. Examples of an array layout are shown in
With the standard curve the concentration of a protein of interest in a sample proceeded in the same way as the calibration reagent can be back calculated.
Therefore, the present invention provides a method for quantifying a protein comprising the epitope of interest in a biological sample comprising
a) immobilizing on an array
b) incubating said array with a detectable affinity reagent of interest,
c) measuring the signal intensity of the bound affinity reagent for each of the two or more concentrations of the calibration reagent and for each of the one or more biological samples,
d) correlating the signal intensity with the amount of epitope of interest, and
e) quantifying the protein comprising the epitope of interest in the one or more biological samples.
The biological sample is of biological origin and a complex mixture of molecules. A sample can be formed e.g. by lysates of cells, cell extracts, body fluids (e.g. whole blood, serum, plasma, urine, tissue fluid, synovial fluid, tears, urine, saliva, and lymph). The samples may be fractioned or non-fractioned.
The biological sample, like the calibration reagent, is applied at the desired position on the array as a spot. The biological samples may be diluted or undiluted with a buffer. In a preferred embodiment said buffer comprises matrix proteins. A preferred matrix protein is BSA, more preferably the matrix protein is acetylated BSA. Typically, the applied samples are allowed to dry before incubating the array with the affinity reagent of interest.
The spots of the biological samples and the calibration reagent on an array are typically arranged in fields. Array fields can form geometrical areas such as e.g. squares, rectangles, circles, and triangles. Examples of an array layout are shown in
Preferably, the applied concentrations of the calibration reagent form a dilution series (e.g. a dilution series of 1:2, 1:5, or 1:10). Also preferred is that the calibration reagent is applied in at least three different concentrations. More preferably, the calibration reagent is applied in 3 to 20 different concentrations, even more preferred are 5 to 15 different concentrations. It is well known by the person skilled in the art how to choose the range of concentrations of the calibration reagent near the expected concentration of the peptide of interest in the biological samples and within the working range of the detection method.
The affinity reagent of interest is incubated at least for 30 minutes on the array, preferably, it is incubated on the array for more than 1 hour, more preferably for 1 to 16 hours, most preferably about 12 hours (12 hours±30 minutes). The excess of affinity reagent is removed and preferably the array is washed before measuring the signal intensity.
Furthermore, the present invention provides the use of the above described standard curve for characterizing the affinity reagent by determining the lower limit of detection, the sensitivity and the dynamic range of the affinity reagent of interest.
The term “lower limit of detection (LOD)” refers to minimum amount of the epitope of interest that can be detected with the affinity reagent. The term “dynamic range” of the affinity reagent refers to the measurable range of concentration of the calibration reagent. The dynamic range is typically determined with a standard curve, whereby the dynamic range is the range of calibration reagent concentrations for which there is a linear or substantially linear correlation to the measured signal. The terms “lower limit of detection” and “dynamic range” are well known to the skilled person in the art.
Therefore, the present invention provides a method for determining the lower limit of detection of an affinity reagent of interest comprising
a) immobilizing the above described calibration reagent in two or more concentrations on an array,
b) incubating said array with an detectable affinity reagent of interest,
c) measuring the signal intensity of the bound affinity reagent for each of the two or more concentrations of the calibration reagent,
d) correlating the signal intensity with amount of epitope of interest, and
e) determining the minimum amount of the epitope of interest that can be detected with the affinity reagent.
In a preferred embodiment, the minimum amount of the detectable epitope of interest can be determined with back-calculating the concentrations which correspond to the signal measured at the blank plus three times the standard deviation of the blank. The blank level is the detected signal of a sample which does not comprise the calibration reagent but is otherwise identical with the samples comprising the calibration reagent.
The calibration reagent is applied as described above. Preferably, the applied two or more concentrations form a dilution series (e.g. a dilution series of 1:2, 1:5, or 1:10). Also preferably, the calibration reagent is applied in at least three different concentrations. More preferably, the calibration reagent is applied in 3 to 20 different concentrations, even more preferred are 5 to 15 different concentrations.
The affinity reagent of interest is incubated at least for 30 minutes on the array, preferably, it is incubated on the array for more than 1 hour, more preferably for 1 to 16 hours, most preferably about 12 hours (12 hours±30 minutes). The excess of affinity reagent is removed and preferably the array is washed before measuring the signal intensity.
The present invention provides a method for determining the sensitivity of the affinity reagent of interest comprising
a) immobilizing the above described calibration reagent in two or more concentrations on an array,
b) incubating said array with a detectable affinity reagent of interest, wherein said affinity reagent of interest,
c) measuring the signal intensity of the bound affinity reagent for each of the two or more concentrations of the calibration reagent,
d) correlating the signal intensity with the amount of epitope of interest, and thereby generating a standard curve,
e) determining the linear part of the standard curve and
f) determining the slope of the linear part of the standard curve.
The calibration reagent is applied as described above. Preferably, the applied two or more concentrations form a dilution series (e.g. a dilution series of 1:2, 1:5, or 1:10). Also preferably, the calibration reagent is applied in at least three different concentrations. More preferably, the calibration reagent is applied in 3 to 20 different concentrations, even more preferred are 5 to 15 different concentrations.
The affinity reagent of interest is incubated at least for 30 minutes on the array, preferably, it is incubated on the array for more than 1 hour, more preferably for 1 to 16 hours, most preferably about 12 hours (12 hours±30 minutes). The excess of affinity reagent is removed and preferably the array is washed before measuring the signal intensity.
The present invention provides a method for determining the dynamic range of the affinity reagent of interest comprising
a) immobilizing the above described calibration reagent in two or more concentrations on an array,
b) incubating said array with a detectable affinity reagent of interest, wherein said affinity reagent of interest,
c) measuring the signal intensity of the bound affinity reagent for each of the two or more concentrations of the calibration reagent,
d) correlating the signal intensity with the amount of epitope of interest, and thereby generating a standard curve,
e) determining the linear part of the standard curve and
f) determining the range of concentration of the calibration reagent of interest of the linear part of the standard curve.
The calibration reagent is applied as described above. Preferably, the applied two or more concentrations form a dilution series (e.g. a dilution series of 1:2, 1:5, or 1:10). Also preferably, the calibration reagent is applied in at least three different concentrations. More preferably, the calibration reagent is applied in 3 to 20 different concentrations, even more preferred are 5 to 15 different concentrations.
The affinity reagent of interest is incubated at least for 30 minutes, preferably it is incubated on the array for more than 1 hour, more preferably for 1 to 16 hours, most preferably about 12 hours (12 hours±30 minutes). The excess of affinity reagent is removed and preferably the array is washed before measuring the signal intensity.
In addition, the calibration reagent can be used for determining the specificity of an affinity reagent. The term “specificity” as used herein refers to the selectivity of the affinity reagent for the epitope of interest. An low specific affinity reagent binds also epitopes other than the epitope of interest.
Therefore, the present invention provides a method for determining the specificity of an affinity reagent comprising the following steps:
a) immobilizing on an array
b) incubating the array with a detectable affinity reagent of interest,
c) measuring the signal intensity of the bound affinity reagent on the array, and
d) comparing the signal intensity correlating with the epitope of interest of the calibration reagent with the signal intensity correlating with the control peptide.
Detection of a significant signal means that the antibody of interest has a low specificity as it recognizes also epitopes other than the epitope of interest. The term “significant signal” as used herein is a signal which is significant higher than the background signal, wherein a background signal is the signal detected in the absence of the a sample (e.g. signal detected between the spots). Significant higher means that the difference to the background signal is statistically relevant (p≦0.05, preferably, p≦0.01).
Preferably, the concentration of the control peptide applied per spot on the array is close (+/−5%) to the concentration of the epitope peptide. Spots of the calibration reagent and spots of samples comprising the control peptide having a similar peptide concentration are preferably grouped on the array in fields, whereby the fields can form geometrical areas like for example squares, rectangles, circles, and triangles. The total protein concentration of the samples in two fields can be different (e.g. a high epitope concentration in field 1 and a low epitope concentration in field 2).
The control epitope does not comprise the epitope of interest, but it comprises an epitope which is different from the epitope of interest. This epitope of the control epitope (control epitope) can for example be the modified equivalent of the epitope of interest (e.g. the unphosphorylated equivalent of the epitope of interest). Preferably, more than one sample comprising a control peptide conjugated to protein carrier is applied on the array. The control peptide in these samples can have different concentrations or they can comprise different epitopes. The control epitope in one sample can for example be the un-phosphorylated equivalent of the epitope of interest and in another sample the control epitope has a different amino acid sequence than the epitope of interest.
The affinity reagent of interest is for at least 30 minutes incubated on the array, preferably for at least 1 hour, more preferably for 1 to 16 hours, most preferably about 12 hours (12 hours±30 minutes). The excess of the mixture is removed and preferably the array is washed before measuring the signal intensity.
Alternatively, the detectable affinity reagent of interest is incubated with a free epitope peptide of interest prior to step a) and in step b) the array is incubated with the mixture of the affinity reagent and free peptide.
Therefore, the present invention also provides a method for determining the specificity of an affinity reagent comprising the following steps:
a) incubating a detectable affinity reagent of interest with free epitope peptide of interest,
b) immobilizing on an array
c) incubating the array with the mixture of the affinity reagent of interest and the free epitope peptide of step a),
d) measuring the signal intensity of the bound affinity reagent on the array and
e) comparing the signal intensity correlating with the epitope of interest of the calibration reagent with the signal intensity correlating with the control peptide.
A “free epitope peptide of interest” is an epitope peptide of interest which is not attached to another molecule. In particular, the free peptide is not attached to a protein carrier.
The concentration of the free peptide is chosen so that the affinity reagent of interest is saturated with the free peptide. This concentration can be determined for example by the following method: a) incubating an detectable affinity reagent of interest with at least to two different concentrations of free epitope peptide, b) immobilizing the above described calibration reagent on at least two arrays, c) incubating the arrays with a mixture of the affinity reagent of interest and the free epitope peptide of step a) wherein each array with a mixture comprising a different concentration of free epitope peptide, d) measuring the signal intensity of the bound affinity reagent on the arrays and determining the concentration of free peptide at which the affinity reagent is saturated with it so that no affinity reagent binds to the array.
The free peptide is preferably incubated with the affinity reagent of interest for at least 30 minutes, preferably for at least 1 hour, more preferably for 1 to 16 hours, most preferably about 12 hours (12 hours±30 minutes).
The mixture of affinity reagent of interest and free peptide is for at least 30 minutes, preferably incubated on the array for at least 1 hour, more preferably for 1 to 16 hours, most preferably about 12 hours (12 hours±30 minutes). The excess of the mixture is removed and preferably the array is washed before measuring the signal intensity.
Having now generally described this invention, the same will become better understood by reference to the specific examples, which are included herein for purpose of illustration only and are not intended to be limiting unless otherwise specified, in connection with the following figures.
for Histone H3 recombinant protein from Upstate).
for Rb recombinant protein from Active Motif).
for Erk1/2 recombinant protein from Invitrogen).
Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated.
Example 1
Lysate Samples
Protein concentrations were determined in a modified Bradford test (Coomassie Plus Protein Assay Reagent, no. 23238, Pierce). The lysate samples were stored in the freezer at −70° C. until use.
For array printing in the different working packages, the lysate samples were adjusted to a given protein concentration in CLB1 (lysis buffer) (Zeptosens) and finally diluted 1:10 in CSBL spotting buffer (Zeptosens)). The final printed protein concentrations are always indicated in the respective sections.
Reverse Phase Protein Microarrays Array Printing
The typical array layout is depicted in
The array was divided into 12 array fields. Each field comprised 12 sample positions, each position printed in duplicate spots. The 12 sample positions were arranged as 3 rows of 4 positions each. 12-point dilutions series (2-fold dilutions) were printed in the order of position 1 (highest concentration=start concentration) to position 12 (lowest concentration), see
Arrays for the different work packages were printed in series of replicates (6 arrays per chip) in a number sufficient to perform all experiments. Print solutions for each series were prepared freshly in 384 well plates by means of a liquid handling robot (Tecan Genesis RSP100). For each standard curve, a stock solution of standard reagent at the start concentration (e.g. 50 nM) was prepared. The different samples (12×2-fold dilutions) were prepared as serial dilutions in the plate wells. The volume per well was 25 μl. For printing of control lysates, samples were adjusted to uniform starting concentration (e.g. 1.5 mg/ml) and diluted 1:10 in spotting buffer CSBL (e.g. final concentration=150 μg/ml).
Each spot was arrayed as a single droplet of about 400 picoliter volume, using a commercial piezo-electric arrayer (NanoPlotter NP2, GeSim GmbH, D-Groβerkmannsdorf). Together with the dilution series and lysate samples, a reference material consisting of fluorescence-labeled protein was co-arrayed into three separate rows of landing marks (see
Samples for dilution series and lysate controls were always prepared freshly from frozen stocks.
After spotting, the microarrays were blocked with BSA, thoroughly washed with ddH2O, dried under a nitrogen stream and stored in the dark at +4° C. until use. For the measurements, a fluidic structure is attached to the chip to address each of the 6 identical arrays of a chip individually with analyte-specific antibody solution at the respective assay condition (the chamber volume per array was about 15 μL).
Antibodies and Assay Reagents
Table 2 lists the proteins and corresponding antibodies used in this study.
NMI-TT provided all other reagents, e.g. labeled detection reagents, buffers, needed to perform the assays on Reverse Phase Protein Arrays (RPA).
Anti-species Fab fragments were used as detection reagents for assay signal generation on the micro arrays.
Assay Buffer (Antibody)
The assay buffer for RPA measurements (assay buffer) was 50 mM imidazole/HCl, 150 mM NaCl, 0.1% Tween20, 0.005% sodium azide, pH7.4 with addition of 5% (w/v) BSA
Print Buffers (Calibration Reagent)
The print buffer was CSBL (Zeptosens—a Division of Bayer Schweiz AG).
The following reagents were used as additions during the study: BSA (#T844.2, Roth)
Reverse Phase Protein Arrays—Assay Procedure and Data Analysis
The detection of the protein analyte on the array was performed in a direct two-step sequential immunoassay. The first step comprised the addition of analyte-specific antibody in assay buffer onto the microarray and incubation for over night at 25° C. After removal of excess antibody by washing with assay buffer, the microarrays were incubated with fluorescence-labeled anti-species Fab fragment for 1 hour at 25° C. in the dark. For the detection of the rabbit antibodies applied in this study, Fab fragments at a 500-fold dilution in assay buffer were used. Finally, the arrays were washed and imaged in solution (assay buffer) with the ZeptoREADER® imager instrument (Zeptosens).
Additional competition experiments were performed to test the specificity of antibody-antigen binding in solution and on the array spot. For this, free synthesized peptide product (specific binding epitope sequences for the respectively applied antibodies) was mixed together with primary antibody in assay buffer solution and incubated for 30 min at room temperature, before the reaction mixture was incubated on the array. All other assay steps were performed at conditions comparable to the normal assay described above. The concentrations of the free peptide were chosen in molar excess of the applied antibody concentration (see also Table 2). The peptide concentrations for competition were typically chosen at 1000 nM, 100 nM and 10 nM, if not stated otherwise.
The ZeptoREADER® is a bench top solution for automatic high throughput readout of microarrays. Shortly, up to 36 microarrays (6 chips) can be mounted into one carrier (MTP footprint format). An integrated stacker allows the unattended readout up to 360 microarrays (10 fully loaded carriers) in a single run. Microarrays can be excited at 532 nm (green) and 635 nm (red); fluorescence emission is detected with emission filters passing between 547-597 nm (green) and 650-700 nm (red). For this study, a series of typically 9 fluorescence images for each array was taken in the red detection channel at exposure times in the range of 0.5-16 seconds and stored in a 16 bit tif format for further analysis with ZeptoVIEW™ PRO software (Zeptosens).
Microarray Analysis
Microarray images were analyzed using the software ZeptoVIEW™ Pro 2.0 (Zeptosens). The spot diameter of the array analysis grid, which was aligned to the microarrays, was set constant at 160 μm.
The data analysis for each measurement was performed as follows:
In addition, Blank assay experiments in the absence of analyte-specific primary antibody were performed to control for possible non-specific binding contributions of the secondary detection reagents. The RFI signals of all blank images were negligible low (for standards as well as lysate samples) and therefore were not considered in the data analysis process.
Data points of the dilution curves (mean signals of duplicate spots) were fitted using the Excel Add-in software package XLfit v4.3.0 (IDBS, Guildford UK). A one-site binding site model was chosen for the fitting (fit function #251: =D+((Vmax*(x̂n))/((x̂n)+(Km̂n))) with D=signal offset, Vmax=saturation signal, Km=affinity constant, n=1 binding site).
Limits-of-Detection (LODs) were as standard concentrations back-calculated from the fit at mean blank signal (4 lowest data points) plus 3-fold standard deviation.
Peptide Sequences
Four antigens were selected to be investigated. These antigens were Histone H3, Rb phosphorylated, Erk1/2 phosphorylated and Erk1. The 4 peptide sequences represent the linear binding epitopes of the 4 selected antigens to respectively chosen antibodies. Epitope sequence information was obtained from the antibody vendors. Antigens, epitope amino acid sequences, lengths, epitope position of the antigens and respective antibody information are summarized in Table 3.
Antibodies against the phosphorylation sites of human Erk1 (p44 MAPK) and Erk2 (p42 MAPK), positioned in the center of the protein, share the same epitope amino acid sequence around amino acid position Thr202 and Tyr204. The antibody against the total form of Erk1 MAPK, here selected from BioSource, was raised against a different linear sequence region positioned at the C-terminal end of the protein. This sequence region is often used also for antibodies of other vendors. The complete peptide sequences of the two proteins Erk1 (SEQ. ID NO: 5) and Erk2 (SEQ. ID NO: 6) are shown in
Peptide Synthesis
For each of the four selected antigens, two peptides have been synthesized at and by NMI-TT. The two peptides comprised (i) a free peptide form to be used as competition reagents in the immunoassays and (ii) a functionalized form to be used for conjugation to BSA proteins as standard reagent molecules on RPA chips. The functionalized peptides were synthesized with a N-terminal Cys-spacer function for covalent coupling to serum albumin protein using capping cycles. Doa-Doa (Doa=8-Amino-3,6-Dioxaoctanoic acid) was chosen as a C18 length equivalent (PEG-like) hydrophilic spacer. Capping cycles were used in the synthesis to achieve a good specificity and final enrichment of the right target sequences for the protein conjugation. After synthesis, peptides were quality controlled by HPLC for good purity, and mass spectrometry (MS) for the correct molecular mass. Sequence information of the synthesized peptide products with corresponding mass information and achieved purity are summarized in Table 4.
All peptides reached a high purity of >95% as specified (most of them >99%) and correct molecular mass. No major difficulties were encountered during the synthesis of these peptides.
Peptide-Protein Conjugation
Final standard reagents were produced as respective peptide-protein conjugates. For this, each functionalized form of the peptide was conjugated in molar excess at 3 different ratios (rations are indicated in Table 5) to pre-activated (maleimide-activated) bovine serum albumin (BSA) via covalent coupling to its free N-terminal Cys group, 2 mg of protein were used for each coupling reaction. The peptide coupling was described earlier in Poetz el al., Proteomics 5, 2402-2411 (2005). Shortly, the solid peptides were dissolved as concentrated stocks in 100% DMSO and were subsequently diluted to working concentrations in PBS pH 7.4 buffer containing DMSO at a maximum of 20%. Peptide and activated BSA solutions were mixed and incubated in the dark for 2 h at room temperature. Unconjugated peptide was removed by means of a spin column (size exclusion) and fractions of the conjugate proteins were collected in PBS pH7.4. Subsequently, the (coupled) peptide concentrations of the peptide-protein fractions were determined by spectrophotometric absorbance measurement at 466 nm (maximum of spectral Dabsyl absorbance, extinction coefficient 33′000 M−1 cm−1, one label per peptide). The absorbance color of the peptide-protein fractions was clearly visible by eye (see
The protein concentrations of the conjugate fractions were determined according to Bradford. Total protein concentrations were around 1.5 mg/ml. Measured peptide concentrations, total protein concentrations and final calculated peptide:protein (dye:protein) ratios of formed products are summarized in Table 5.
10 x
2.7x
0.7x
Quality control of the peptide conjugates was performed via SDS-PAGE (4-12% gel) using the pure pre-activated BSA as a reference. Gels were Coomassie stained for 60 min. The gel images showed pure product bands as expected, with mass shifts corresponding to the different calculated peptide:protein coupling ratios. Typically, finally determined coupling ratios reached 17-40% of the initially prepared molar excess ratios of peptide:protein, which was according to our previous experience with other peptides. The variations may be due to different solubilities at the applied high starting concentrations and/or e.g. different peptide conformational structures of the peptides in the aqueous coupling buffer.
All four peptide-protein conjugate standard reagents were pure according to PAGE.
Finally, all peptide reagents were lyophilized: min. 5 mg of each free peptide (4 competitor reagents), and min. 1 mg of each peptide-protein conjugate (4 standard reagents, at selected peptide:protein ratio).
Recombinant Proteins and SDS-PAGE Control (Histone, Erk)
8 recombinant proteins from different vendors were mutually selected as full protein alternatives to peptide standards (Table 6). 7 proteins (2× Histone H3, 5× Erk) were quality controlled by SDS-PAGE using BSA as a reference proteins.
The purity of the proteins was good as evident from the single bands after the gel electrophoresis. However, signal instensities of the single bands showed large differences indicating different concentrations of the protein when compared to same amounts of BSA loaded as a reference. Obviously values of concentrations given in the data sheets of the vendors were not reliable. Therefore, the integral signal intensities of the protein bands were analyzed to estimate at best the right concentrations relative to co-loaded BSA. Resulting correction factors (see Table 6) were therefore considered in the sample preparation of all standard dilution series printed in the following.
Optimization of Assay and Print Conditions—First Standard Curves
In first experiments, assays were performed on different sets of arrays which were printed with standard curves of the different peptide-BSA reagents at different compositions and conditions, to examine their effects on subsequently tested immunoassay performance. The following conditions were examined:
Standard curves were printed as 12 serial dilutions curves (2-fold dilutions), each dilution as duplicate spots (as described in Example 1: Material and Methods). Start concentrations of the different reagents for printing were adjusted to a uniform epitope concentration of 50 nM. In addition, positive and negative control lysates were co-printed into same arrays. The lysate samples were arrayed at a total protein concentration of 0.25 mg/ml. Immunoassays were performed at the antibody conditions indicated. Observed differences on assay performance were evaluated qualitatively and, based on these results and previous experience with these types of reagents, best print and assay conditions were selected.
Standard Curves of Peptide Reagents Containing Different Peptide:Protein Conjugate Ratios
Generally, standard curves of printed peptide standard reagents at different peptide:protein ratios provided almost comparable signals in the assays. Assay images are depicted in
Dynamic Range of Signals and Concentrations
The assays on the printed standard curves demonstrated very prominent signals and a high dynamic range of signals which can be extracted from one and the same measurement in one image.
The start concentrations of these first standard curves printed was chosen at 50 nM. In a signal comparison to the co-printed control lysate samples, it turned out that assays signals of the standard curves and hence the highest start concentration of standards were much higher than the intrinsic values (levels) of the respective control lysates, especially for the phophorylated protein analytes. Therefore start concentrations of standard curves had to be adjusted respectively. Also the signal differences of negative and positive control lysates, respectively expression levels, were very low, especially for the phosphorylated analytes pErk and pRb (obviously positive treatment of the prepared cell lines had been suboptimal). Therefore new control lysates were prepared and provided (see Table 1).
Standard Curves of Peptide-BSA Reagents in Absence/Presence of Matrix Protein Additions
In another set of arrays, standard curves of peptide standard reagents and first recombinant proteins were printed at three different buffer conditions: (i) in the absence of any additional protein addition, and in the presence of (ii) 50 μg/ml an (iii) 100 μg/ml matrix protein (acetylated BSA=acBSA), as depicted in
Summary of best selected print and assay conditions for this study:
Uniform print condition selected for all standard curves:
Spotting buffer CSBL plus addition of 50 μg/ml acetylated BSA (acBSA)
Start concentrations selected for dilution series (peptide standards):
10 nM Histone H3
1 nM pRb
2.5 nM pERk1/2
5 nM ERk1/2
Assay conditions (antibody dilutions) selected:
1:10000 for Histone H3 assay
1:250 for pRb assay
1:500 for pErk1/2 assay
1:1000 for Erk1/2 assays (3 antibodies)
Signals of printed reference spots were adjusted to typically 15000 gray levels at 4 s image exposure time.
Arrays were printed with standard curves of the 4 peptide standard reagents (HistoneH3-BSA 2.7×, pRb-BSA 1×, pErk-BSA 2.7× and Erk1-BSA 2.7×) as well as all available recombinant proteins for comparison (12 standard curves with 12 point dilution curves). Control lysate sample controls (negative and positive controls, new delivery samples) were co-printed at a total protein concentration of 400 μg/ml and 250 μg/ml. Standards and control lysates were prepared in spotting buffer CSBL, standards with additions of 50 μg/ml acBSA. Start concentrations of the standard curve samples were adjusted to reach in minimum the assay signals of the positive control lysates. Array layout and conditions are summarized in Table 7.
Assay were performed on the arrays for each of the four protein analytes in the absence (normal assay) and presence of increasing concentrations of corresponding free peptide, which was pre-mixed with the respective antibody solution before incubation on the arrays (competition assays). Typically three different concentrations of free peptide (1000 nM, 100 nM and 10nM, if not otherwise indicated) were tested for their efficiency to complex with respective antibody to suppress the formation of specific antibody-protein analyte complexes on the array spots. In addition, competition assay were performed with antibody solutions which were pre-incubated with corresponding recombinant proteins, to compare their competition efficiency and specificity with that of the free peptide reagents. Blank assays (in absence of primary antibody) were performed as additional controls but their signals were negligibly low and therefore were not considered in the quantitative data analysis.
Tables 8 to 11 summarize the quantitative results in terms of maximum standard curve signals.
The results of the Erk1/2 assays nicely demonstrated that not only the Erk1/2 antibody form Biosource, but also the two additionally chosen CST antibodies (#4695 rb monoclonal and #9102 rb polyclonal) specifically recognized only the Erk1-BSA standard spots (and at comparable signal intensities), but not the pErk-BSA standard spots. This implies that all three antibodies from the three different vendors used in this project obviously were raised against a very similar peptide motif at the C-terminal end of the protein. This was further corroborated by an additional earlier competition experiment (add-on experiment), which was performed with the Erk1/2 antibody form CST (#9102) in the presence of a free epitope peptide which represented the amino acid sequence of the Erk1/2 phosphorylation site (as used in this project) but was not phosphorylated (de-phospho peptide, available at NMI). In the competition assay, performed under otherwise comparable conditions as was shown before, this de-phospho peptide was not able to suppress the specific signals of the standard and lysate spots observed in the respective Erk1/2 normal assay (data not shown). It is therefore shown that the Erk antibodies purchased from CST use different protein epitope sequences to differentiate between the total and phosphorylated forms of the Erk1/2 protein.
Histone
0.322
0.010
0.127
0.080
0.088
1.68
0.228
3.93
0.267
H3-BSA 2.7x
Histone H3 Roche
0.015
<0.01
0.001
0.004
0.004
0.027
2.56
0.214
2.50
0.074
Histone H3 Upstate
11.18
0.195
0.007
0.031
0.155
0.115
2.09
0.062
3.16
0.269
pRb-BSA 1x
0.47
0.019
<0.01
0.001
<0.01
0.003
0.07
0.003
pRb Active Motif
0.04
0.002
<0.01
0.000
<0.01
0.000
<0.01
0.001
pErk-BSA 2.7x
3.47
0.092
0.10
0.018
0.45
0.024
1.05
0.062
pErk Active Motif
0.19
0.019
0.01
0.006
0.01
0.002
0.01
0.001
pErk1 Invitrogen
1.66
0.050
0.01
0.001
0.01
0.004
0.05
0.007
Erk1 Invitrogen
10.91
0.409
0.02
0.001
0.38
0.014
5.17
0.056
Erk1-BSA 2.7x
8.24
0.356
0.14
0.018
5.69
0.327
7.88
0.210
pErk Active Motif
1.17
0.129
0.01
0.000
0.15
0.003
0.31
0.007
pErk1 Invitrogen
10.43
0.372
0.02
0.003
0.32
0.026
3.88
0.010
Erk2 Biosource
3.57
0.085
0.01
0.001
0.18
0.005
0.90
0.095
Erk1 CST
0.28
0.017
<0.01
0.001
0.16
0.004
0.15
0.002
Erk1 Invitrogen
7.12
0.152
0.04
0.001
0.59
0.006
no assay
—
Erk1-BSA 2.7x
2.77
0.159
0.01
0.002
0.29
0.015
no assay
—
pErk Active Motif
0.22
0.052
<0.01
0.000
0.11
0.001
no assay
—
pErk1 Invitrogen
5.17
0.373
0.02
0.000
0.39
0.003
no assay
—
Erk2 Biosource
1.51
0.072
0.01
0.001
0.16
0.002
no assay
—
Erk1 CST
0.04
0.003
<0.01
0.002
0.10
0.001
no assay
—
Erk1 Invitrogen
8.96
0.047
7.28
0.221
0.19
0.000
no assay
—
Erk1-BSA 2.7x
2.44
0.002
0.06
0.000
0.12
0.004
no assay
—
pErk Active Motif
0.36
0.041
0.23
0.007
0.03
0.004
no assay
—
pErk1 Invitrogen
7.20
0.261
4.65
0.049
0.11
0.001
no assay
—
Erk2 Biosource
1.51
0.222
0.91
0.034
0.07
0.003
no assay
—
Erk1 CST
0.05
0.000
0.03
0.003
0.03
0.001
no assay
—
All assays were performed on arrays of the same layout as shown in
Assays were performed for each of the four protein analytes in the absence (normal assay) and presence of free peptide at the highest concentration effective for complete competition (competition assays). Each condition (normal assay, competition assay) was measured in duplicate assays (two arrays per condition). Blank assays (in absence of primary antibody) were additionally measured as a control. All array images were analyzed quantitatively. For each assay, standard signal curves for each of the 12 array fields of each array were generated by fitting a one-site binding model to the data points extracted from each of the printed 12-point dilution series. Limits-of-detection (LOD) were determined from the fit curve as back-calculated concentrations which corresponded to the mean signals at blank levels (four lowest data points) plus 3-fold respective standard deviations.
Generated standard curves of the normal and competition assays (data points and fitting curves, as well as back-calculated LOD values) for the duplicate assays are shown in the
Abcam antibody specifically bound to HistoneH3-BSA peptide standard and the Abcam antibody specifically bound also to Histone H3 recombinant proteins, most prominently to the human protein from Upstate. Signal intensities of standard curves of peptide standard and recombinant protein (Upstate) were well comparable. Reproducibilities of the two assays were very good. Signal CVs were typically about 12% for the peptide standards and about 13% for Histone H3 protein (Upsate). The mean LODs were 0.123±0.019 nM for peptide standard, and 0.156±0.023 nM for recombinant protein (Upstate). LOD values were well reproducible for the duplicate assays and comparable for peptide standard and protein (
The CST antibody specifically bbound to pRb-BSA peptide standard and the CST antibody specifically bound also to pRb recombinant protein from Active Motif. However the signal intensities of the protein standard curves were clearly lower and reach only about 10% .of the peptide standard curves. We presume that the protein is not or only partly phosphorylated (note: pRb and Rb annotation in public data banks is obviously used in parallel for the same protein and it was not clear to us whether pRb used here indicated the phosphorylated protein). Reproducibilities of the two assays were very good. Signal CVs were typically about 7% for the peptide standards and slightly higher at about 12% for pRb protein. LOD values were well reproducible for the duplicate assays. The mean LODs were 0.025±0.001 nM for peptide standard, and 0.097±0.020 nM for recombinant protein (
The CST antibody specifically bound only to pErk1/2-BSA peptide standard, and not to Erk1-BSA standard. The CST antibody specifically bound also prominently to the pErk1 recombinant protein (Invitrogen) and reaches signal intensities of about 25% of the respective pErk1/2-BSA peptide standard signals. The CST antibody bound to a minor degree also to pErk from Active Motif (about 12%) ≧Erk1 from CST (about 11%) ≧Erk1 protein from Invitrogen (about 3%). Signals are given relative to the signal of pErk1 protein (Invitrogen) in %. Reproducibilities of the two assays were very good. Signal CVs were typically about 2% for the peptide standard, and slightly higher at about 6% for the pErk1 protein (Invitrogen). LOD values were well reproducible for the duplicate assays. The mean LODs were 0.030±0.002 nM for peptide standard, and 0.055±0.003 nM for pErk1 protein (Invitrogen) (
Good quality of fit curves achieved with correlation coefficients of r2>0.99
Biosource antibody specifically bound only to Erk1-BSA peptide standard, not to pErk1-BSA standard. Biosource antibody specifically bound to Erk1 and pErk1 recombinant proteins (most prominently among the different proteins available), and generated well comparable signal intensities for these proteins and Erk1-BSA peptide standards. Biosource antibody bound to a lower degree also to Erk2 protein (Biosource)>pErk (Active Motif)>Erk1 (CST). Reproducibilities of the two assays were very good. Signal CVs were typically about 3% for the peptide standard, and slightly higher about 8% for Erk1 protein and about 5%for pErk1 protein. LOD values were well reproducible for the duplicate assays. The mean LODs were 0.046±0.001 nM for peptide standard, and 0.072±0.013 nM for recombinant Erk1 protein (Invitrogen), and 0.044±0.004 nM for recombinant pErk1protein (Invitrogen) (
Good quality of fit curves achieved with correlation coefficients of r2>0.99
Determination of Absolute Protein Analyte Concentrations
Assays were performed on arrays of the layout shown in the following
Duplicate assays (on 2 arrays) were performed for each of the four protein analytes. Blank assays (in absence of primary antibody) were additionally measured as a control. All array images were analyzed quantitatively. For each assay, standard signal curves for each of the 6 array fields of each array were generated by fitting a one-site binding model to the data points extracted from each of the printed 8-point dilution series. Data points were averaged for the maximum number of replicate spots available in each series (N=5 or N=10). For a comparison, mean signals were also calculated for duplicate spots (center rows of each field). Limits-of-detection (LODs) were determined from the fit curves as described before. In addition, signals of spike-in series were corrected for the endogenous (blank) signals and corrected signal were projected into the standard curves in buffer.
Results are shown in
Histone H3 peptide: The assays revealed the specific signal response for dilutions curves of the Histone H3 peptide standard. Blank assay showed zero response. Signals of lysate spiked with Histone H3 followed the signals of the standard curve in buffer at comparable, but slightly lower offset intensities (after subtraction of endogenous Histone H3 signal level of the pure lysate). Reproducibilities of the two assays were very good. Endogenous concentration of Histone H3 protein in pure lysate was determined by back-calculation of the blank signals of lysate from standard curve fits. The mean concentration was 0.063±0.005 nM. Other lysate spots showed marginally low signals (
pRB assay: The assays revealed the specific signal response for dilution curves of the pRb peptide standard. Blank assay showed zero response. Signals of lysate spiked with pRb followed the signals of the standard curve in buffer at comparable, but slightly lower offset intensities (after subtraction of endogenous pRb signal level of the pure lysate). Reproducibilities of the two assays were very good. Endogenous concentration of pRb protein in pure lysate was determined by back-calculation of the blank signals of lysate from the standard curve fits. The mean concentration of endogenous protein was 0.066±0.010 nM. Other spots containing lysate 6 (negative for Histone H3) and lysate 13 (negative for pErk1/2) showed constant signals at different intensity which obviously represent their endogenous levels of pRb protein in these lysates. Good quality of fit curves achieved with correlation coefficients of r2>0.99 (
pERK1/2 assay (CST #9101): The assays revealed the specific signal response for dilution curves of the pErk1/2 peptide standard. Blank assay showed zero response. Signals of lysate spiked with pErk1/2 followed the signals of the standard curve in buffer at comparable, but slightly higher offset intensities (after subtraction of endogenous pErk signal level of the pure lysate). Reproducibilities of the two assays were very good. Endogenous concentration of pErk1/2 protein in pure lysate was determined by back-calculation of the blank signals of lysate from the standard curve fits. The mean concentration of endogenous protein was 0.149±0.005 nM. Other spots containing lysate 6 (negative for Histone H3) and lysate 12 (negative for pRb) showed constant signals at different intensity which obviously represent their endogenous levels of pErk1/2 protein in these lysates. Good quality of fit curves achieved with correlation coefficients of r2>0.99 (
pERK1/2 assay (BioSource 44-654G): The assays revealed no signal response for dilution curves of all applied peptide standards, as expected. Blank assay showed zero response. All spots containing lysates showed constant signals at different intensity which obviously represent their endogenous levels of Erk1/2 protein in these lysates.
General Remark to Effect of Increased Number of Replicate Spots:
For all 4 assays, analyzed data were compared for the effect of number of replicate spots on coefficients of variations (CVs). Mean signals of all analyte-specific signals were formed from all available number of replicate spot signals (N=5 or N=10 per condition) and from N=2 replicate spot signals (chosen form the center rows in each array field). In almost all cases, the CVs of the duplicate spot analysis were comparable or slightly smaller than for the N=5 or N=10 replicate spot analysis (to mention that this was observed at a persistently low level of CVs throughout all experiments).
Advantages of peptide standard reagents: composition of the molecules (peptide to protein ratios) can be well prepared in a reproducible manner- concentration/number of epitope sequences per molecules could be well determined by the introduction of a small absorbance label (Dabsyl) in each peptide sequence. No adverse effects of the label were observed in the RPA assays degree of phsophorylation of synthetic peptide standards is well determined and 100% In contrary, the degree of phosphorylation of commercial recombinant protein preparations is probably largely variable and not easy to determine (see our results with several protein candidates of different vendors, for the case of Erk/pErk) peptide-protein conjugates use BSA as uniform carrier protein (BSA is a well characterized molecule for protein array applications). We expect signal responses of different epitope peptide standards not largely impacted by the (same) carrier protein properties.
In contrary, we have measured prominent differences in assay signal response from recombinant proteins of the different vendors (e.g for the case of Erk), which might be also due to the different protein preparations and characteristics (different expression systems, ±GST-tag, ±His-tag etc.)
All 4 free forms of synthesized peptides achieved complete competition of peptide standard signals. There was a trend that phospho-peptides reached the full competition at lower concentrations which may indicative for higher affinities of the applied antibodies to phospho-epitopes. We observed no major impact or adverse effects of the competitor peptide on assay response or array quality.
In contrary, recombinant proteins used as competitors generated additional signal background on the arrays (partly by factors higher than the specific spot signals e.g. for the case of Histone H3 proteins) which made the array analysis difficult or even impossible. Nevertheless, also the recombinant proteins seemed to suppress the signals of the original standard curves. However, the recombinant proteins could not suppress the signals of the control lysates, but even generated additional signals on the lysate spots which might be due to non-specific binding to other proteins in the lysates. This implies that peptides are clearly preferable as competitor reagents.
On the lysate spots, competition with peptides could lead to complete suppression of lysate signals (e.g. for Histone H3 lysates), but also to partial (not complete) suppression leaving a basal signal even at the highest concentrations of competitor applied (e.g for pRb, pErk lysates) which might be due to a certain non-specific binding contribution of the applied antibodies. Therefore using competition and normal assays in parallel might be proposed as a universal concept to measure all future analytes-of-interest.
Standard curves generated form the dilution series printed on the RPA were generally of high quality which manifested in low CVs of replicate spots signals and good fits to data points with correlation coefficients of r2>0.99 in all cases. Standard curves of peptide reagents showed the trend for better fit correlations (smaller r2 values) than standard curves of recombinant proteins.
Signal CVs of duplicate spots (N=2) and of increased number of replicate spots (N=5, N=10) were comparable indicating that standard curves from printed duplicate spots already provided optimum results.
Reproducibility of duplicate assay were also very good as manifested in low CVs of mean standard signals (array-to-array), which were in the range of a few to 10 percent. Standard curves of peptide reagents showed the trend for lower CVs (mean CV=6%) than for recombinant proteins (mean CV=9%)
Signal intensities of standard curves of the 2 total protein analytes (Histone H3 and Erk1) matched very good. Standard curves of the 2 phosphorylated protein analytes (pRb and pErk) showed lower signals for the recombinant proteins, probably due to a lower and less defined degree of phopshorylation
The figures depict the array images of the duplicate assays and the graphs of the peptide standard curves in buffer, curves of the peptide standards spiked into respective lysate and combined curves of peptide standards in buffer and spiked into lysate after correction of endogenous protein concentrations of the pure lysates.
| Number | Date | Country | Kind |
|---|---|---|---|
| 09176130.4 | Nov 2009 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP10/67379 | 11/12/2010 | WO | 00 | 5/15/2012 |
