Post-translational modifications of FDA-approved plasma biomarkers in glioblastoma samples.

Liquid chromatography-tandem mass spectrometry was used to analyze plasma proteins of volunteers (control) and patients with glioblastoma multiform (GBM). A database search was pre-set with a variable post-translational modification (PTM): phosphorylation, acetylation or ubiquitination. There were no significant differences between the control and the GBM groups regarding the number of protein identifications, sequence coverage or number of PTMs.

Plasma preparation to measure FDA-approved protein markers by selected reaction monitoring.

Background: The development of commercially available panels for human blood plasma screening via selected reaction monitoring (SRM) offers reliable, cost-efficient and highly-standardized discovery and validation of protein biomarkers. However, protein detection by SRM can be hampered by interfering peptide fragment ions. To estimate the influence of interference on protein detection, we performed different types of sample preparation and implemented SRM measurements for well-characterized protein targets approved by the US Food and Drug Administration.

One-dimensional proteomic profiling of Danio rerio embryo vitellogenin to estimate quantum dot toxicity.

Background: Vitellogenin (Vtg) is the major egg yolk protein (YP) in most oviparous species and may be useful as an indicator in ecotoxicological testing at the biochemical level. In this study, we obtained detailed information about the Vtgs of Danio rerio embryos by cutting SDS-PAGE gel lanes into thin slices, and analyzing them slice-by-slice with (MALDI-TOF) mass spectrometry.

Results: We conducted three proteomic analyses, comparing embryonic Danio rerio Vtg cleavage products after exposure for 48 h to CdSecore/ZnSshell quantum dots (QDs), after exposure to a mixture of the components used for quantum dot synthesis (MCS-QDs), and in untreated embryos.

Applying of hierarchical clustering to analysis of protein patterns in the human cancer-associated liver.

Background: There are two ways that statistical methods can learn from biomedical data. One way is to learn classifiers to identify diseases and to predict outcomes using the training dataset with established diagnosis for each sample. When the training dataset is not available the task can be to mine for presence of meaningful groups (clusters) of samples and to explore underlying data structure (unsupervised learning).

Producing a one-dimensional proteomic map for human liver cytochromes p450.

In this chapter we explore the inducible cytochrome P450 (CYP) forms as an example of membrane proteins analysis that relies on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) fractionation with subsequent mass spectrometric (MS) identification. The approach involves cutting an SDS-PAGE gel lane into thin slices and identifying proteins in each slice by MS with the aim of obtaining detailed information on proteins of interest. A one-dimensional proteomic map showing the distribution of selected CYP isoforms across 40 slices was constructed using mass spectra obtained from each slice.

Computational approach to characterization of human liver drug-metabolizing enzymes.

Cytochromes P450 are the key enzymes for activating and inactivating many drugs; individual expression levels of CYPs may play a crucial role in drug safety and drug efficacy. Statistical comparison of biochemical profiles of 23 human liver microsomes have been used to characterize human liver samples. The profile included 12 parameters, namely activity of NADPH-cytochrome P450 reductase, cytochrome P450 content and cytochrome P450-dependent monooxygenase activities with marker substrates.

Application of slicing of one-dimensional gels with subsequent slice-by-slice mass spectrometry for the proteomic profiling of human liver cytochromes P450.

Sequential thin slicing of one-dimensional electrophoresis gels followed by slice-by-slice mass spectrometry to allow protein identification was used to produce a proteomic map for cytochromes P450. Parallel MALDI-TOF-MS and LC-MS/MS analyses were performed. Combination of the two MS methods increased the quality of protein identification.

Fluorescent assay for riboflavin binding to cytochrome P450 2B4.

The interactions between the hemoprotein cytochrome P450 2B4 (CYP 2B4) and riboflavin - a low molecular weight component of the flavoprotein NADPH-dependent cytochrome P450 reductase - were investigated by fluorescence spectroscopy. Riboflavin fluorescence quenching by cytochrome P450 2B4 was used to probe the ligand-enzyme binding (lambda(ex)=385 nm, lambda(em)=520 nm). Fluorescence titration experiments showed formation of a complex between cytochrome P450 2B4 and riboflavin with an apparent dissociation constant value, K(d)=8.