Collection and storage of the clinical samples are crucial factors in the metabolomic workflows. However, with the expansion of metabolomics into the clinical domain and towards the large field ...studies in particular, the high sampling/storage standards practiced in the tightly controlled hospital environment cannot always be guaranteed. Thus, if the samples are exposed to suboptimal conditions and their integrity is compromised should they be discarded? Or such samples retain physiologically relevant information and can be of use? To explore the options we analyzed 117 urine samples that were collected under two different conditions. Part of the samples were collected within a clinical setting under optimal conditions, another part by patients at home and shipped to the hospital by mail. All samples were analyzed by liquid chromatography–mass spectrometry (LC–MS) and proton nuclear magnetic resonance (¹H NMR) spectroscopy. Multivariate modelling revealed clear differences between the two sampling conditions for both LC–MS and¹H NMR data sets. However, the differential metabolites appeared to be platform-specific, which clearly emphasizes the complementary nature of both techniques. The analysis of the samples that were exposed to suboptimal conditions revealed that age and body mass index remain as dominant traits of the metabolic profile, although their influence was stronger for LC–MS data. In conclusion, although it is important to ensure adequate sample collection and storage conditions, urine samples that do not fulfil these criteria still retain valuable physiological information and as such thus they could be of use for metabolomic studies when no alternative is available.
Urine is potentially a rich source of peptide biomarkers, but reproducible, high-throughput peptidomic analysis is often hampered by the inherent variability in factors such as pH and salt ...concentration. Our goal was to develop a generally applicable, rapid, and robust method for screening large numbers of urine samples, resulting in a broad spectrum of native peptides, as a tool to be used for biomarker discovery.
Peptide samples were trapped, desalted, pH-normalized, and fractionated on a miniaturized automatic reverse-phase strong cation exchange (RP-SCX) cartridge system. We analyzed eluted peptides using MALDI-TOF, Fourier transform ion cyclotron resonance, and liquid chromatography-iontrap mass spectrometry. We determined qualitative and quantitative reproducibility of the system and robustness of the method using BSA digests and urine samples, and we used a selected set of urine samples from Schistosoma haematobium-infected individuals to evaluate clinical applicability.
The automated RP-SCX sample cleanup and fractionation system exhibits a high qualitative and quantitative reproducibility, with both BSA standards and urine samples. Because of the relatively high cartridge binding capacity (1-2 mL urine), eluted peptides can be measured with high sensitivity using multiple mass spectrometric techniques. As proof of principle, hemoglobin-derived peptides were identified in urine samples from S. haematobium-infected individuals, even when the microhematuria test was negative.
We present a practical, step-by-step method for screening and identification of urinary peptides. Alongside the analytical method evaluation on standard samples, we demonstrate its feasibility with actual clinical material.
Urine represents the most easily obtainable body fluid and consequently one of the most common samples in clinical chemistry. The majority of pathological changes in human organs may well be ...reflected in urine. In this way, urine analysis can aid in disease diagnosis, treatment monitoring, and prognosis. Currently, the most commonly used method for identification of new urine biomarkers involves centrifugation of the urine sample to collect either the soluble urine proteins or the urinary exosomes followed by 1 or 2 protein purification and separation steps before visualization and finally identification of potential biomarkers, usually by mass spectrometry. Here we present a generally applicable, rapid, and robust method for screening large number of urine samples, resulting in a broad spectrum of native peptides, as a tool to be used for biomarker discovery. The method combines online sample pretreatment with a well-established mass spectrometric technique. Native peptides are extracted from urine samples on a miniaturized reverse-phase-strong cation exchange cartridge system. As the proper identification of native peptides often requires combination of data acquired on different mass analyzers, we have aimed at a procedure providing us with sufficient material to identify and characterize the differentially expressed markers.
Imbalances in the amounts of amyloid-beta peptides (A beta) generated by the membrane proteases beta- and gamma-secretase are considered as a trigger of Alzheimer's disease (AD). Cell-free studies of ...gamma-secretase have shown that increasing membrane thickness modulates A beta generation but it has remained unclear if these effects are translatable to cells. Here we show that the very long-chain fatty acid erucic acid (EA) triggers acyl chain remodeling in AD cell models, resulting in substantial lipidome alterations which included increased esterification of EA in membrane lipids. Membrane remodeling enhanced gamma-secretase processivity, resulting in the increased production of the potentially beneficial A beta 37 and/or A beta 38 species in multiple cell lines. Unexpectedly, we found that the membrane remodeling stimulated total A beta secretion by cells expressing WT gamma-secre-tase but lowered it for cells expressing an aggressive familial AD mutant gamma-secretase. We conclude that EA-mediated mod-ulation of membrane composition is accompanied by complex lipid homeostatic changes that can impact amyloidogenic processing in different ways and elicit distinct gamma-secretase re-sponses, providing critical implications for lipid-based AD treatment strategies.
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