Identification of in vitro protein biomarkers of idiosyncratic liver toxicity

In Vitro Biomarker Discovery for Atherosclerosis by Proteomics

“…The use of an immortalized cell line, rather than ex vivo-derived macrophages, was also necessary because of the number of cells required to achieve the high level of total protein required for the various processing steps leading up to MS while remaining above the lower limit of detection. A previous study had demonstrated the ability of gel-free multidimensional LC, coupled to MS/MS, to rapidly and effectively analyze cellular supernatants for proteins that change in abundance as a result of treatment (29).…”

“…Aliquots of oxidized LDL and LDL were previously analyzed by this method to determine their retention times (data not shown). Fractions containing the secreted/excreted proteins, but not the LDL or oxidized LDL, were reduced, alkylated, and digested as described previously (29). The digests were subjected to size-exclusion chromatography (G2000SW XL ; Tosoh) in order to remove undigested protein and excess trypsin (30).…”

Guidelines for the Routine Application of the Peptide Hits Technique

“…T he use of protein biomarkers to detect diseases in their earliest stages holds the potential to save thousands of lives [1]. The advancement of proteomic techniques has recently led to the discovery of a series of biomarkers for different disease states [2][3][4][5][6]. While the traditional proteomic method (two-dimensional gel electrophoresis (2D-PAGE) followed by mass spectrometry) to elucidate differences between normal and disease-state samples still remains in use, this technique is not adequately reproducible, labor intensive, and hard to automate [7,8].…”

“…Although much less precise than the labeling methods, this PHT is much easier to implement and therefore less prone to errors or experimental artifacts. The PHT has been proven successful in several biomarker and target discovery programs [2,3,5,19]. Liu et al and Blondeau et al have used a similar concept named spectral sampling and successfully estimated relative protein abundance in complex protein mixtures [20 -22].…”

“…Previous projects using the PHT demonstrated that statistical tools commonly used for gene array experiments were also applicable to proteomic data [3,18]. The purpose of the project described here was to refine, optimize, and guide the application of these bioinformatic techniques by analyzing a well-defined sample.…”