What does it need to be a biomarker? Relationships between resolution, differential quantification and statistical validation of protein surrogate biomarkers

Abstract

The separation of proteins with the aim of discovering surrogate biomarkers defining differences between various stages of biological materials is the core occupation of every project in Proteomics. There are numerous recent publications suggesting a wide array of separation technologies, ranging from 2-DE, MS-linked LC, CE or chip-based surface-enhanced laser desorption ionization claiming to be useful for this purpose, and addressing the urgent clinical, diagnostic or toxicological needs for such surrogates. However, many potential biomarkers emerging from proteomic studies did not survive validation in, for example, large-scale clinical studies or simply independent experiments, and at the same time being tested in settings with case numbers bigger than perhaps a few hundreds. The major problems of protein biomarkers are associated with the huge dynamic range of possible concentrations and the ever-increasing number of molecular species due to post-translational modifications. In particular, the chemical diversity of the latter imposes a necessity of improved resolution of separation technologies, because otherwise the crucial quantitative information is lost in pools of poorly resolved peptides. Here, we present and analyze some examples of successful developments of protein biomarkers, and show the prerequisites and necessary considerations while moving protein candidates from purely descriptive phenomena to a stage of validated surrogate biomarkers. This includes a detailed discussion of requirements regarding resolution of initial separation techniques, linear dynamic range and statistics of differential quantification, but also the subsequent clinical validation, testing the biomarker in clinical settings and using large numbers of patient samples.

The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation

“…In brief, small amounts of each sample were labelled with 125 I and 131 I for differential pattern control. the signals from these two isotopes were used for statistical treatment of abundance differences (Schrattenholz and Groebe, 2007). Spots were analysed first with a high throughput peptide mass fingerprint procedure based on MAlDI-tOF-MS. For those spots for which no unambiguous identification was achieved a fragment ion analysis based on lC-eSI-Iontrap-MS/MS was added Groebe et al, 2007).…”

Protein Expression Profiling in Esophageal Adenocarcinoma Patients Indicates Association of Heat-Shock Protein 27 Expression and Chemotherapy Response

“…A differential quantification of potential cancer markers has to consider the resolution of the analytic procedure and dynamic range of the detection method. For complex samples, radioactive labeling and high-resolution two-dimensional electrophoresis offer nearly the only alternative to obtain quantitative data that can be treated in the necessary statistical way (14,23). The approach has been successfully applied for the detection of differentially expressed proteins in cancer and nonneoplastic tissue of the prostate (16), breast (15), and kidney (30).…”

“…Radioactive images of preparative gels were warped to the same target as the ProteoTope images through the Delta2D software package by DECO-DON. 10 The above labels were transferred to the corresponding silverstained images of the preparative gels, and the spot coordinates of the labeled spots were exported as pick lists for automatic excision and processing as described (14,23).…”

“…Proteomic analysis has emerged as a valuable tool in scientific medicine (11 -13), albeit with considerable quantitative and statistical challenges (14). Because the chemoresistance of tumors is a complex and multifactorial event, we did differential quantitative protein expression analysis in an explorative setting by using the recently developed and established radioactive ProteoTope clinical proteomics platform (15 -17).…”

Nutriproteomics: technologies and applications for identification and quantification of biomarkers and ingredients

“…estimated 10 12 in human blood) (14) . Current MS-based proteomic platforms can deliver a dynamic range of 10 4 . This means that the low-abundant proteome has to be addressed by depletion of the most abundant proteins (15) or by selective enrichment of low-abundant proteins (16)(17)(18) .…”