Experimental Design for SELDI
Successful experimental biomarker study design requires selection of the appropriate proteomics platform. One platform for biomarker discovery is surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), which combines the separation power of chromatography and high-sensitivity mass spectrometry.
SELDI technology meets the biomarker discovery challenge by providing the sensitivity required to reproducibly detect low-abundance proteins in the presence of many high-abundance ones, while also providing the throughput required to analyze the enormous number of samples required to validate candidate biomarkers and ensure their clinical utility.
The ProteinChip® SELDI system from Bio-Rad Laboratories (www.bio-rad.com) utilizes arrays that selectively bind and retain whole classes of proteins from complex samples. The mass profile of the bound proteins is then determined directly from the arrays using TOF MS, creating protein profiles of molecular mass versus peak intensity. SELDI is one of the few platforms that can analyze hundreds of proteins in thousands of samples in a timeframe commensurate with the demands of a clinical proteomics biomarker study.
As with any technique applied to biomarker studies, successful application of the ProteinChip SELDI system for discovery requires careful experimental design. The design should include optimization of procedures used for sample preparation, selection and processing of arrays, data acquisition with a focus on optimizing laser energy, and data analysis. Different array types and wash conditions generate different profiles from the same sample, and combining these conditions yields a much broader picture of the proteome (Figure 3).
Sample preparation is a potential source of analytical bias that is often overlooked and underemphasized. Consistent and appropriate liquid-handling techniques as well as defined protocols for the initial processing of samples are essential for obtaining reproducible results. Sample types such as serum and plasma are highly complex, and though addition of any sample-handling step increases chances for variability, fractionation of these sample types prior to SELDI analysis increases the number of protein peaks detected and improves detection of low-abundance proteins.
Array processing is also a key to success with SELDI, and careful thought should go into sample layout on each array, optimizing sample dilution and buffer composition and standardizing the methods for application of the matrix to the arrays. Array preparation for a single condition (one fraction, array chemistry, and matrix combination) and data collection on this condition should be completed before continuing to the next condition.
Proper data collection and analysis are essential to successful biomarker discovery with SELDI. Qualification and calibration of a SELDI-TOF MS system should be done regularly to ensure optimum performance; the manufacturer provides kits for this purpose. Data-acquisition parameters should be tested and optimized on a pool of experimental samples before collecting data from study samples.
The collected data can first be processed using the system’s default processing parameters, then reprocessed later if necessary. Statistical tests are generally used to screen for peaks that show significant differences between clinically relevant groups using either univariate or multivariate statistical techniques, and care should be taken to avoid false discovery and overfitting of multivariate models.
The application of proteomics to discover clinically meaningful biomarkers has proven to be challenging and has so far met with only limited success. However, the combination of a coherent, rigorous, and comprehensive process from study design to clinical assay implementation with the ProteinChip SELDI technology will help meet the challenge of discovering biomarker panels that could be used to accurately detect and predict human disease states, customize disease treatment, and assist in all phases of drug development.