With regard to the selected methodology, for MS-based peptide profiling approaches the problems can be categorized as follows. First of all, multiple profiling studies

have shown to selleck chemicals lack reproducibility and could not be validated. In this context, standardization of the protocols used for serum sample collection and for peptide and protein purification is pivotal [10], [13] and [14]. The use of a fully automated high-throughput platform for sample processing based on solid-phase extraction (SPE) has been shown to minimize variation and to improve robustness of the method [15]. Secondly, previous MS-acquisitions such as performed on surface-enhanced laser desorption/ionization (SELDI) platforms were not robust and yielded poor accuracies. In addition, identification of peptides or proteins was cumbersome, or not possible at all in these early profiling studies. However, with current equipment these issues can be considered obsolete. buy AZD6738 The use of internal standards in combination with modern mass analyzers now allows precise quantitation and detailed characterization of peptides in high-throughput profiles [16] and [17]. Thirdly, similar peptide profiles were found for various diseases, implying that the features

were not specific. On the other hand, it has been postulated that well-defined degradation of highly abundant proteins into peptides (“degradome”) Sorafenib order can result in tumor-specific serum peptidome patterns [18]. Recently, we reported a protein profiling

study for PC performed on a fully automated SPE-based serum processing platform [19]. Proteins were first isolated with weak cation exchange (WCX) magnetic beads (MBs) using a 96-channel liquid handling robot, followed by acquisition of linear mode MALDI-TOF profiles in the range of 1 to 12 kDa, and evaluation via linear discriminant analysis with double cross-validation. This resulted in a discriminating WCX-profile for PC with a sensitivity of 78% and a specificity of 89% in the calibration set with an area under the curve (AUC) of 90%. These results were validated with a sensitivity of 74% and a specificity of 91% (AUC 90%). However, an obvious disadvantage of low resolution MS profiles is the fact that (poly)peptides and proteins are measured as broad peaks, thus leading to one of the earlier mentioned problems on peak identification. In a second profiling study using the same PC cohort, serum samples were processed with reversed-phase (RP) C18 MBs, and resulting peptides were measured with high resolution reflectron mode MALDI-TOF MS yielding isotopically resolved profiles up to 4 kDa. For statistical evaluation, a list of 42 different peptides was compiled from which a discriminating profile for PC could be defined, with an area under the curve (AUC) of 92% (98%) a sensitivity of 76% (95%) and specificity of 91% (100%) in the calibration (validation) set.