Prediction of high-responding peptides for targeted protein assays by mass spectrometry

Abstract

Protein biomarker discovery produces lengthy lists of candidates that must subsequently be verified in blood or other accessible biofluids. Use of targeted mass spectrometry (MS) to verify disease-or therapy-related changes in protein levels requires the selection of peptides that are quantifiable surrogates for proteins of interest. Peptides that produce the highest ion-current response (highresponding peptides) are likely to provide the best detection sensitivity. Identification of the most effective signature peptides, particularly in the absence of experimental data, remains a major resource constraint in developing targeted MS-based assays. Here we describe a computational method that uses protein physicochemical properties to select high-responding peptides and demonstrate its utility in identifying signature peptides in plasma, a complex proteome with a wide range of protein concentrations. Our method, which employs a Random Forest classifier, facilitates the development of targeted MS-based assays for biomarker verification or any application where protein levels need to be measured.Proteomic discovery experiments in case-and-control comparisons of tissue or proximal fluids frequently generate lists comprising many tens to hundreds of candidate biomarkers 1 . Integrative genomic approaches incorporating microarray data and literature mining are also increasingly being used to guide identification of candidate protein biomarkers. To further credential biomarker candidates and move them toward possible clinical implementation, it is necessary to determine which of the proteins from lists of candidates differentially abundant in diseased versus healthy patients can be detected in body fluids, such as blood, that can be assayed with minimal invasiveness 1 .This process, termed verification, has historically been approached using antibodies. Highquality, well-characterized collections of antibodies suitable for protein detection in tissue are now being developed 2 . But unfortunately, the required immunoassay-grade antibody pairs necessary for sensitive and specific detection in blood exist for only a tiny percentage of the proteome. Thus, for the majority of proteins, suitable reagents for their detection and quantification in blood (or other biofluids) do not yet exist and alternative technologies are needed to bridge the gap between discovery and clinical-assay development. This problem is NIH Public Access Author ManuscriptNat Biotechnol. Author manuscript; available in PMC 2009 September 28. Published in final edited form as:Nat Biotechnol. 2009 February ; 27(2): 190-198. doi:10.1038/nbt.1524. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptan important aspect of the larger need in biology and medicine for quantitative methods to measure the presence and abundance of any protein of interest.Targeted MS is emerging as an assay technology capable of selective and sensitive detection and quantification of potentially any protein of interest (or modification thereof) in the ...

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