Serum Microarrays for Large Scale Screening of Protein Levels

Abstract

There is a great need for comprehensive proteomic analysis of large patient cohorts of plasma and serum samples to identify biomarkers of human diseases. Here we describe a new antibody-based proteomic approach involving a reverse array format where serum samples are spotted on a microarray. This enables all samples to be screened for their content of a certain serum protein in a single experiment using target-recognizing antibodies and fluorescently labeled secondary antibodies. The procedure is illustrated with the analysis of the IgA levels in 2009 spotted serum samples, and the data are compared with clinical routine measurements. The results suggest that it is possible to simultaneously screen thousands of complex clinical serum samples for their content of the relative amount of specific serum proteins of clinical relevance. Molecular & Cellular Proteomics 4:1942-1947, 2005.The development of DNA microarrays has had an enormous impact in the research field of functional genomics where it has enabled large scale global analysis of whole genomes and transcriptomes. The developed methodology and technology have now become an established and relatively mature technique in terms of available instrumentation for production and analysis as well as commercially available premade microarrays, robust experimental protocols, and the long range of available tools and software for data analysis. Naturally there have been large efforts to continue the development of the microarray format with similar approaches for global protein analysis where great potential can be envisioned.The microarray technology makes it possible to analyze a large number of proteins within a small sample volume in a single experiment or with a reverse set-up analyze a limited number of proteins in many samples. The basic principles for highly sensitive "microspot" ligand binding assays were described by Ekins (1) and Ekins and Chu (2), who showed that small amounts of capture molecules in microspots can detect low concentrations of the analyte with high accuracy and sensitivity.The protein microarray applications can be divided into two categories: functional protein arrays and protein profiling arrays. Functional protein arrays can effectively screen large quantities of proteins for biochemical activity, protein-protein interactions, protein-lipid interactions, protein-nucleic acid interactions, and protein-small molecule interactions. The arrays have been used to study activity of uncharacterized proteins (3), antibody specificity profiling (4), and immune response profiling. Protein abundance arrays are used to measure protein abundance and/or alterations (5). Lately there has been progress in microarrays printed with antigen and used for detection of circulating antibodies in clinical specimens (6 -8). The protein profiling microarrays have also been used to measure the binding specificity of protein expression libraries (9, 10) and for protein profiling in cancer tissue (11).Currently there are two main types of profiling microarrays: forw...

Human body fluid proteome analysis

“…Other array formats used for serum protein analysis include selfassembled monolayer array, filtration-based array, multiplexed photoaptamer-based arrays, phage-based array, and RP array [135][136][137][138][139]. The filtration-based protein array can improve the overall reaction kinetic rate by ten-fold and enhance the sensitivity and specificity of the assay.…”

“…The filtration-based protein array can improve the overall reaction kinetic rate by ten-fold and enhance the sensitivity and specificity of the assay. In RP array, serum samples are spotted on a microarray and screened for their content of a specific serum protein in a single experiment using target-recognizing antibodies and fluorescently labeled secondary antibodies [137]. The photoaptamer-based arrays utilize photoaptamers covalently bind to their target analytes before fluorescent signal detection.…”

Advances and Challenges in Liquid Chromatography-Mass Spectrometry-based Proteomics Profiling for Clinical Applications

“…Many different technologies have been applied for biomarker discovery and other clinical applications, including twodimensional (2D) gel-electrophoresis (6), LC-MS, and proteinand antibody-based microarrays (7)(8)(9). LC-MS-or tandem MS (MS/MS)-based proteomics technologies offer highly sensitive analytical capabilities and a relatively large dynamic range of detection and have increasingly become the method of choice for in depth profiling of complex protein mixtures (1).…”

Antibody Arrays: Technical Considerations and Clinical Applications in Cancer

“…Antibody arrays for bladder cancer have been designed by selecting antibodies against targets differentially expressed in bladder tumors identified by gene profiling (40 ). Serum protein profiles obtained by 2 independent antibody arrays provided comprehensive tools for bladder cancer diagnosis and clinical outcome stratification (40 ). Validation analyses with ELISA and immunohistochemistry on tissue microarrays confirmed the relevance of identified proteins for tumor progression.…”

“…Reverse protein arrays have also been optimized for use with spot serum specimens and to obtain high-throughput measurement of IgA in thousands of sera from a single experiment (39 ). Antibody arrays for bladder cancer have been designed by selecting antibodies against targets differentially expressed in bladder tumors identified by gene profiling (40 ). Serum protein profiles obtained by 2 independent antibody arrays provided comprehensive tools for bladder cancer diagnosis and clinical outcome stratification (40 ).…”