Targeted proteomic strategy for clinical biomarker discovery

Abstract

The high complexity and large dynamic range of blood plasma proteins currently prohibit the sensitive and high-throughput profiling of disease and control plasma proteome sample sets large enough to reliably detect disease indicating differences. To circumvent these technological limitations we describe here a new two-stage strategy for the mass spectrometry (MS) assisted discovery, verification and validation of disease biomarkers. In an initial discovery phase N-linked glycoproteins with distinguishable expression patterns in primary normal and diseased tissue are detected and identified. In the second step the proteins identified in the initial phase are subjected to targeted MS analysis in plasma samples, using the highly sensitive and specific selected reaction monitoring (SRM) technology. Since glycosylated proteins, such as those secreted or shed from the cell surface are likely to reside and persist in blood, the two-stage strategy is focused on the quantification of tissue derived glycoproteins in plasma. The focus on the N-glycoproteome not only reduces the complexity of the analytes, but also targets an information-rich subproteome which is relevant for remote sensing of diseases in the plasma. The N-glycoprotein based biomarker discovery and validation workflow reviewed here allows for the robust identification of protein candidate panels that can finally be selectively monitored in the blood plasma at high sensitivity in a reliable, non-invasive and quantitative fashion.

Figure 1

Depicted are the plasma protein concentration as described by Anderson and Anderson (2002). The proteins can be grouped in three main categories (classical plasma proteins, tissue leakage products, interleukins/cytokines). Red dots indicate proteins that were identified by the HUPO plasma proteome initiative (States et al., 2006) and yellow dots represent currently utilized biomarkers (Polanski and Anderson, 2006).

Figure 2

Schematic diagram of analysis of N‐glycosites from tissues/cells and plasma. Cell surface proteins and secreted proteins from tissues/cells and plasma are processed using glycopeptide capture method, glyco‐peptides are analyzed by mass spectrometry and identified by SEQUEST search. The identified peptides and proteins from tissues/cells and plasma are compared and the tissue/cell specific proteins are identified. Figure was adapted from Zhang et al. (2007).

Figure 3

Scheme for biomarker discovery, qualification, verification and validation. Solid phase enrichment of N‐glycopeptides (SPEG) can be performed to discover in vivo disease‐specific signatures using cells, tissue and finally blood plasma and MS‐based label‐free quantification. The selected reaction monitoring (SRM) assays of these protein panel are then qualified and later verified in human patients by SRM and eventually validated again by SRM or ELISA.