Quantitative Profiling

During normal cellular metabolism reactive oxygen species (ROS) are inevitably formed as by-products of respiration. ROS are extremely reactive molecules and can react with and damage surrounding DNA, protein and lipid molecules and subsequently alter their normal function in the cell. This oxidative damage is counter balanced by antioxidant and repair mechanisms, which are present in every aerobic cell. When the levels of oxidative damage increase or the antioxidant and repair capacity is inefficient to protect against this damage a state of oxidative stress occurs.

Cellular systems are essential model systems to study reactive oxygen species and oxidative damage but there are widely accepted technical difficulties with available methods for quantifying endogenous oxidative damage in these systems. Here we present a stable isotope dilution UPLC-MS/MS protocol for measuring F2-isoprostanes as accurate markers for endogenous oxidative damage in cellular systems. F2-isoprostanes are chemically stable prostaglandin-like lipid peroxidation products of arachidonic acid, the predominant polyunsaturated fatty acid in mammalian cells.

Accumulation of oxidative damage has been proposed to be causal to aging as defined by the Free radical Theory of Aging, which has been subject to recent debate. However, a major hurdle in understanding the biological roles of reactive oxygen species (ROS) signaling and their oxidative damage has been the widely recognized methodological difficulties to measure oxidative damage and ROS in vivo.

BACKGROUND:

Analysis of sialic acid (SA) metabolites in cerebrospinal fluid (CSF) is important for clinical diagnosis. In the present study, a high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) method for free sialic acid (FSA) and total sialic acid (TSA) in human CSF was validated.

Ceramides (CERs), cholesterol, and free fatty acids (FFAs) are the main lipid classes in human stratum corneum (SC, outermost skin layer), but no studies report on the detailed analysis of these classes in a single platform. The primary aims of this study were to 1) develop an LC/MS method for (semi-)quantitative analysis of all main lipid classes present in human SC; and 2) use this method to study in detail the lipid profiles of human skin substitutes and compare them to human SC lipids.

Because of its high sensitivity and specificity, hyphenated mass spectrometry has become the predominant method to detect and quantify metabolites present in bio-samples relevant for all sorts of life science studies being executed. In contrast to targeted methods that are dedicated to specific features, global profiling acquisition methods allow new unspecific metabolites to be analyzed. The challenge with these so-called untargeted methods is the proper and automated extraction and integration of features that could be of relevance.

The application of CE-MS in the field of metabolomics is underrepresented, even though it is in principle highly suited for the analysis of small charged compounds, as many metabolites are. Moreover, a robust coupling, using the sheath-liquid (SL) assisted interface was already presented more than a decade ago. A lack of concentration sensitivity is often mentioned as a reason for the underrepresentation of CE-MS in metabolomics. This is caused by postcolumn dilution of the sample with SL, which is typically delivered at a flow rate of 1-10 μL/min.