Pan, S. et al. High throughput proteome screening for biomarker detection. Mol. Cell. Proteomics 4, 182-190

Institute for Systems Biology, Seattle, WA 98103, USA.
Molecular &amp Cellular Proteomics (Impact Factor: 6.56). 03/2005; 4(2):182-90. DOI: 10.1074/mcp.M400161-MCP200
Source: PubMed


Mass spectrometry-based quantitative proteomics has become an important component of biological and clinical research. Current methods, while highly developed and powerful, are falling short of their goal of routinely analyzing whole proteomes mainly because the wealth of proteomic information accumulated from prior studies is not used for the planning or interpretation of present experiments. The consequence of this situation is that in every proteomic experiment the proteome is rediscovered. In this report we describe an approach for quantitative proteomics that builds on the extensive prior knowledge of proteomes and a platform for the implementation of the method. The method is based on the selection and chemical synthesis of isotopically labeled reference peptides that uniquely identify a particular protein and the addition of a panel of such peptides to the sample mixture consisting of tryptic peptides from the proteome in question. The platform consists of a peptide separation module for the generation of ordered peptide arrays from the combined peptide sample on the sample plate of a MALDI mass spectrometer, a high throughput MALDI-TOF/TOF mass spectrometer, and a suite of software tools for the selective analysis of the targeted peptides and the interpretation of the results. Applying the method to the analysis of the human blood serum proteome we demonstrate the feasibility of using mass spectrometry-based proteomics as a high throughput screening technology for the detection and quantification of targeted proteins in a complex system.

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    • "The relative quantification needs efficient labeling of the detected peptides or proteins and also relies on the accuracy of the mass measurement and the chromatographic reproducibility . In contrast to the relative quantification strategies just described, the absolute quantification of proteins is attractive in many studies, such as discovery of new biomarkers in vast clinical samples (Pan et al., 2005). When the absolute quantification of proteins is known in different samples, then their relative ratios can be calculated readily. "
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    • "However, analysis of these fluids pose limitations to studying disease-associated proteins because of the wide dynamic range of proteins and abundance of common proteins in complex biological samples. These limitations, in part, explain the inability to discover low abundance serum proteins [35] [36] [37]. A more focused approach would be to deconstruct CICs as the key contributors to pathology. "
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    • "The major difficulty associated with these approaches centers on the availability of specific antibodies against novel proteins identified by proteomics and the difficulty to detect changes of low abundant proteins/peptides by these methods. To circumvent this problem, several groups of investigators are actively involved in MS-based targeted quantitative proteomics (Aebersold, 2003; Anderson, 2005; Gerber et al., 2003; Pan et al., 2005). This technology uses isotope dilution followed by MS analysis, in which test-samples are supplemented (spiked) with isotope-labeled synthetic peptides, which serve as the signature markers for the identification and quantification of native peptides (target) within each sample. "
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