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Systems biology

Vol.22no.102006,pages1278–1279

doi:10.1093/bioinformatics/btl093 BIOINFORMATICS APPLICATIONS NOTE

Systems biology

ProMAT:protein microarray analysis tool

Amanda M.White1,?,Don S.Daly1,Susan M.Varnum2,Kevin K.Anderson3,

Nikki Bollinger2and Richard C.Zangar2

1Statistical Sciences,2Cell Biology and Biochemistry and3Decision and Sensor Analytics,

Paci?c Northwest National Laboratory,Richland,WA99354,USA

Received on December20,2005;revised on March7,2006;accepted on March9,2006

Advance Access publication April4,2006

Associate Editor:Martin Bishop

ABSTRACT

Summary:ProMAT is a software tool for statistically analyzing data from enzyme-linked immunosorbent assay microarray experiments. The software estimates standard curves,sample protein concentra-tions and their uncertainties for multiple assays.ProMAT generates a set of comprehensive figures for assessing results and diagnosing pro-cess quality.The tool is available for Windows or Mac,and is distributed as open-source Java and R code.

Availability:ProMAT is available at https://www.wendangku.net/doc/b018320939.html,/statistics/ ProMAT.ProMAT requires Java version1.5.0and R version1.9.1 (or more recent versions).ProMAT requires either Windows XP or Mac OS10.4or newer versions.

Contact:amanda.white@https://www.wendangku.net/doc/b018320939.html,

INTRODUCTION

Enzyme-linked immunosorbent assays(ELISA)are widely used to estimate the concentration of a speci?c protein.Proteomic techno-logies have recently increased the rate by which protein biomarker candidates are being discovered,such that traditional ELISA approaches(e.g.96-well plates)have become an inef?cient means to validate biomarkers.ELISA in a microarray format per-mits simultaneous estimation of the concentrations of numerous proteins in a small sample and therefore can increase the rate of biomarker validation.High-throughput analysis of ELISA microar-rays requires statistical software speci?cally designed for estimating standard curves,predicting protein concentrations and estimating their uncertainties(Zangar et al.,2006).Currently,there are no freeware tools suitable for this use.Typically,researchers are forced to do these calculations using an inef?cient spreadsheet program which is not designed for these calculations.Therefore,we have developed a statistically based bioinformatics tool,ProMAT,which signi?cantly decreases ELISA microarray data analysis time while increasing the information content of the results. SOFTWARE CAPABILITIES

ELISA microarray experiments consist of two component analyses that are undertaken in parallel.One component is the analysis of a serial dilution of protein standards.This analysis is used to estimate the standard curves which model protein concentration as a function of spot?uorescence.The second component is the analysis of the biological samples.The protein concentrations in the biological samples are estimated from their corresponding standard curves. These concentration estimates,however,are inherently uncertain because of the uncertainty in both the estimates of the standard curve and the?uorescence intensity of the biological samples. Evaluating this uncertainty is critical for interpreting the data and optimizing experimental procedures.Therefore,estimation of uncertainty has been automated in ProMAT.The statistical methods used to?t standard curves and estimate uncertainties are discussed in depth by Daly and co-workers(Daly et al.,2005).

For each antigen,multiple statistical models are?t to the standard data,and the PRESS statistic(Myers,1990)is used to select the best ?tting model.Currently,the four-parameter logistic model spotIntensity?A+

BàA

1+exp

and the power curve model

spotIntensity?m·concentration n+b

are used,although additional models may be incorporated in future. The models are?t using‘nlm’,a nonlinear optimization procedure in R,which also provides uncertainty estimates for the curve para-meters(e.g.A,B,m,n).Replicate spots on the biological sample arrays are used to estimate the uncertainty of the spot intensities. Then,upper and lower bounds on the sample concentration predic-tions are estimated by incorporating the uncertainties in both the model parameter and biological spot intensity estimates through propagation of error(Mood et al.,1974).

ProMAT combines these elements into a simple but comprehens-ive diagnostic?gure(Fig.1)that allows the user to quickly assess the assay quality.This?gure shows statistical summaries of both standard and sample data for a single antigen.The lower right panel shows the standard data for this antigen(black points)along with the estimated standard curve(black line).The upper and lower blue lines in this panel are the$95%con?dence bounds on the concentration prediction.The red dotted lines delineate a prediction acceptance region;sample data points that fall within this region are considered to be high-quality data.The lower left panel shows a histogram of sample spot intensities on a vertical axis aligned with that of the standard curve.The top panel shows the percent coef?cient of variation

%CV?100·

predicted concentration

To whom correspondence should be addressed.

1278óThe Author2006.Published by Oxford University Press.All rights reserved.For Permissions,please email:journals.permissions@https://www.wendangku.net/doc/b018320939.html,

for each point along the standard curve.This diagnostic ?gure allows users to quickly identify problems such as the sample signal intensities being outside the acceptable region of the curve,high variability in the standard data,or a standard curve that has a limited useful range (i.e.red lines are close together).

ProMAT takes as input the tables of spot intensity values output by most microarray image analysis tools,along with information about the slide layout (e.g.the antigen corresponding to each spot)and standard dilution series.ProMAT output is in the form of ASCII tables in comma-delimited format showing the con-centration predictions and $95%bounds for each sample-antigen-array combination.Diagnostic ?gures of the type shown

in Figure 1are produced for each antigen,and an HTML page is created to allow the user to easily browse a complete set of these ?gures.

SOFTWARE IMPLEMENTATION

ProMAT will run under Windows XP or Mac OS 10.4or newer versions.ProMAT’s user-interface is written in Java;the underlying statistical algorithms are written in R (https://www.wendangku.net/doc/b018320939.html,),an open-source statistical programming language.ProMAT is distrib-uted with its source code so that it may be adapted or customized if desired.

CONCLUSIONS

ProMAT is a tool for the statistical analysis of ELISA microarray experiment datasets.ProMAT estimates standard curves,sample protein concentrations and their uncertainties,and it provides insightful diagnostic ?gures.This is the ?rst open-source tool speci?cally designed for protein microarrays.ProMAT runs on Windows or Mac operating systems.

ACKNOWLEDGEMENTS

Funding for this work was provided by the US Department of Energy through the Laboratory Directed Research and Development pro-gram and by the National Cancer Institute and the Early Detection Research Network (CA117378).Pacific Northwest National Laboratory is operated by Battelle for the US Department of Energy under Contract DE-AC06-76RL01830.Conflict of Interest:none declared.

REFERENCES

Daly,D.S.et al.(2005)Evaluating concentration estimation errors in ELISA microarray

experiments.BMC Bioinformatics ,6,17.

Mood,A.M.,Graybill,F.A.and Boes,D.C.(1974)Introduction to the Theory of

Statistics .McGraw-Hill Book Company,New York.

Myers,R.H.(1990)Classical and Modern Regression with Applications .

PWS Publishing Co,Boston.

Zangar,R.C.et al.(2006)ELISA Microarray technology into a high-throughput system

for cancer biomarker validation.Expert Rev.Proteomics ,3,

37–44.

Fig.1.A representative ProMAT diagnostic figure from the ELISA micro-array standards and sample data for a single assay.ProMAT generates a similar figure for each assay in an ELISA microarray experiment.

ProMAT

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Scene 1: Accommodation 住宿
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