Development of a protein chip: a MS-based method for quantitation of protein expression and modification levels using an immunoaffinity approach.

Abstract

Protein chip technology permits analysis of the expression and modification status of numerous targeted proteins within a single experiment, mainly through the use of antibody-based microarrays. Despite recent improvements in these protein chips, their applications are still limited for a variety of reasons, which include technical challenges in fabrication of the antibody chips as well as the very low specificity achieved by current detection methods. We have developed a unique approach for relative and/or absolute quantitation of protein expression and modification based on the capture of epitope peptides on affinity beads, which can be used to develop a mass-spectrometry-based protein chip technology. This new method, which utilizes antibodies immobilized on beads for the capture of target peptides, instead of proteins, eliminates many of the problems previously associated with protein chips. We present here several proof-of-principle experiments examining model peptides by this technique. These experiments show that the method is capable of (i). detecting peptides bound to a single antibody bead, (ii). detecting peptides at low (fmol) levels, (iii). producing MS/MS data of suitable quality for protein identification via database searching or de novo sequencing, (iv). quantitating peptides affinity-bound to antibody beads, (v). specifically detecting target peptides in complex mixtures over wide dynamic ranges, and (vi) is compatible with a microarray format for high-throughput analysis. Because our novel method uses antibody beads instead of a derivatized capture surface, and peptides instead of proteins for affinity capture, it can overcome many of the pitfalls of previous protein chip fabrications. Therefore, this method offers an improved approach to protein chip technology that should prove useful for diagnostics and drug development applications.