MALDI imaging MS reveals candidate lipid markers of polycystic kidney disease.

Autosomal recessive polycystic kidney disease (ARPKD) is a severe, monogenetically inherited kidney and liver disease. PCK rats carrying the orthologous mutant gene serve as a model of human disease, and alterations in lipid profiles in PCK rats suggest that defined subsets of lipids may be useful as molecular disease markers. Whereas MALDI protein imaging mass spectrometry (IMS) has become a promising tool for disease classification, widely applicable workflows that link MALDI lipid imaging and identification as well as structural characterization of candidate disease-classifying marker lipids are lacking. Here, we combine selective MALDI imaging of sulfated kidney lipids and Fisher discriminant analysis (FDA) of imaging data sets for identification of candidate markers of progressive disease in PCK rats. Our study highlights strong increases in lower mass lipids as main classifiers of cystic disease. Structure determination by high-resolution mass spectrometry identifies these altered lipids as taurine-conjugated bile acids. These sulfated lipids are selectively elevated in the PCK rat model but not in models of related hepatorenal fibrocystic diseases, suggesting that they be molecular markers of the disease and that a combination of MALDI imaging with high-resolution MS methods and Fisher discriminant data analysis may be applicable for lipid marker discovery.

Quantitative characterization of tissue globotetraosylceramides in a rat model of polycystic kidney disease by PrimaDrop sample preparation and indirect high-performance thin layer chromatography-matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry with automated data acquisition.

Glycosphingolipids (GSL) have been associated with a variety of diseases, including cancer and autosomal dominant polycystic kidney disease (ADPKD). In contrast to glucosylceramide and gangliosides, alterations in complex neutral GSLs such as globotetraosylceramide (Gb4Cer) have not been investigated in ADPKD yet, and mass spectrometry analysis of Gb4Cer from tissue extracts remains challenging. To this end, we introduce PrimaDrop as an improved and widely applicable sample preparation method for automated matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis of lipid extracts, which promotes homogeneous cocrystallization and enables relative quantification by indirect thin layer chromatography (TLC)-MALDI-time-of-flight (TOF)-MS against an internal bradykinin standard. Application of the method for detailed investigation of Gb4Cer isoforms in kidneys of an ADPKD rat model revealed increased levels of sphingoid base-containing isoforms in cystic kidneys, whereas changes were subtle for Gb4Cer-containing phytosphingoid bases. We furthermore established an absolute LC-ESI-MS/MS quantification method and demonstrate that absolute quantities of Gb4Cer correlate well with relative quantities obtained by indirect TLC-MALDI-TOF-MS. Taken together, our study proposes an effective sample preparation method for automated analysis of lipid extracts and TLC eluates and suggests that indirect high-performace (HP)TLC-MALDI-TOF-MS with automated data acquisition is a viable option for analysis of neutral glycosphingolipids and that Gb4Cer may play a role in the pathogenesis of ADPKD.

Peripheral profiling analysis for bipolar disorder reveals markers associated with reduced cell survival.

Little is known about the molecular factors that are altered in remitting bipolar disorder (BD) patients. We carried out proteome profiling of peripheral blood mononuclear cells (PBMCs) and serum from BD patients who were not experiencing mania or major depression (euthymia) compared to matched healthy controls using liquid chromatography-mass spectrometry (LC-MS(E) ) and Multi-Analyte Profiling (Human Map(®) ) platforms. This resulted in the identification of approximately 60 differentially expressed molecules involved predominantly in cell death/survival pathways. In PBMCs, this was manifested in cytoskeletal and stress response-associated proteins, whereas most serum analytes were associated with the inflammatory response. The predicted effect of serum analytes on physiological systems was tested by treating PBMCs with serum obtained from the same patients, resulting in reduced cellular survival. These preliminary results suggest that BD patients carry a peripheral fingerprint that has detrimental effects on cell function and that could be used to distinguish BD patients from healthy controls despite being in a remission phase. It is hoped that additional studies of BD patients in the manic and depressed stages could lead to the identification of a molecular fingerprint that could be used for predicting episodic switching and for guiding treatment strategies.

Myotubularin-related protein 7 inhibits insulin signaling in colorectal cancer.

Phosphoinositide (PIP) phosphatases such as myotubularins (MTMs) inhibit growth factor receptor signaling. However, the function of myotubularin-related protein 7 (MTMR7) in cancer is unknown. We show that MTMR7 protein was down-regulated with increasing tumor grade (G), size (T) and stage (UICC) in patients with colorectal cancer (CRC) (n=1786). The presence of MTMR7 in the stroma correlated with poor prognosis, whereas MTMR7 expression in the tumor was not predictive for patients' survival. Insulin reduced MTMR7 protein levels in human CRC cell lines, and CRC patients with type 2 diabetes mellitus (T2DM) or loss of imprinting (LOI) of insulin-like growth factor 2 (IGF2) had an increased risk for MTMR7 loss. Mechanistically, MTMR7 lowered PIPs and inhibited insulin-mediated AKT-ERK1/2 signaling and proliferation in human CRC cell lines. MTMR7 provides a novel link between growth factor signaling and cancer, and may thus constitute a potential marker or drug target for human CRC.

Identification of serum proteins bound to industrial nanomaterials.

Nanoparticles (NPs) are decorated with proteins and other biomolecules when they get into contact with biological systems. The presence of proteins in cell culture medium can therefore have effects on the biological outcome in cell-based tests. In this study, the manufactured nanomaterials silicon dioxide (SiO(2)), titanium dioxide (TiO(2)), iron-III-oxide (Fe(2)O(3)), and carbon black (CB) were used to study their interaction with single proteins from bovine and human plasma (albumin, fibrinogen and IgG) as well as with complete human serum. The protein binding capacity of the material was investigated and 1D gel electrophoresis was used to separate the bound proteins and to identify the bands by matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF) mass spectrometry. We found that the NP surface chemistry had a great impact on the amount of bound protein with distinct ligands for each of the tested particles. The hydrophobic CB NPs bound much more protein than the hydrophilic metal oxide NPs. Among the single proteins investigated, fibrinogen showed the strongest affinity for SiO(2), TiO(2) and CB NPs. The identified proteins from human serum adsorbed to these NPs were very different. Only apolipoprotein A1 was found to be adsorbed to all NPs. These studies will help to explain the different degree of biological responses observed after in vitro exposure of cells in the absence or presence of serum and might also support the interpretation of in vivo experiments were NPs come directly into contact with blood plasma.