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Proteomic-basierende Identifikation von Biomarkern für akute und chronische Nierentransplantatabstossung. (68421)
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  • Open Access
    Stefan Schaub; John A. Wilkins; Mihaela Antonovici; Oleg V. Krokhin; Tracey Weiler; David N. Rush; Peter Nickerson;
    Project: SNSF | Proteomic-basierende Iden... (68421)

    Our aim is to develop noninvasive tests to monitor the renal allograft posttransplant. Previously we have reported that an unbiased proteomic based approach can detect urine protein peaks associated with acute tubulointerstitial renal allograft rejection. Identification of these proteins peaks by mass spectrometry demonstrated that they all derive from nontryptic cleaved forms of beta2 microglobulin. In vitro experiments showed that cleavage of intact beta2 microglobulin requires a urine pH < 6 and the presence of aspartic proteases. Patients with acute tubulointerstitial rejection had lower urine pH than stable transplants and healthy individuals. In addition they had higher amounts of aspartic proteases and intact beta2 microglobulin in urine. These factors ultimately lead to increased amounts of cleaved urinary beta2 microglobulin. Cleaved beta2 microglobulin as an indicator of acute tubular injury may become a useful tool for noninvasive monitoring of renal allografts.

  • Open Access
    Stefan Schaub; John A. Wilkins; Tracey Weiler; Kevin Sangster; David N. Rush; Peter Nickerson;
    Publisher: Elsevier BV
    Project: CIHR , SNSF | Proteomic-basierende Iden... (68421)

    Urine protein profiling with surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry. Background In the last few years there has been an increasing interest in exploring the human proteome. In particular, efforts have focused on developing strategies to generate reproducible protein maps of normal cells, tissues, and biologic fluids, from which studies can then compare protein expression between different groups (e.g., healthy individuals vs. those with a specific pathologic state). Methods Various extrinsic factors (instrument settings, matrix composition, urine storage post void, freeze-thaw cycles) and intrinsic factors (blood in urine, urine dilution, first-void vs. midstream urine) were analyzed with respect to their impact on urine protein profiling using surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Results Extrinsic factors that critically influenced reproducibility and peak detection of urine protein profiling were matrix composition and instrument settings, while freeze-thaw cycles had minimal impact. Midstream urines samples did not undergo changes in their protein profile when stored for three days at 4°C. Intrinsic factors that influenced normal urine protein profiling were blood in the urine and urine dilution. Female first-void urine had a significantly different ratio of proteins present compared to a midstream urine sample. Limitations of the SELDI-TOF-MS technique included ion suppression and quantification of individual proteins when protein composition was complex. Conclusion SELDI-TOF-MS offers a unique platform for high throughput urine protein profiling; however, standardization of analysis conditions is critical, and both extrinsic and intrinsic factors must be taken into account for accurate data interpretation.

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