title: Potential of UCHL1 as biomarker for destruction of pancreatic beta cells.
abstract: There is a clinical need for plasma tests for real-time detection of beta cell destruction, as surrogate endpoint in islet transplantation and immunoprevention trials in type 1 diabetes. This study reports on the use of label-free LC-MS/MS proteomics for bottom-up selection of candidate biomarkers. Ubiquitin COOH-terminal hydrolase 1 (UCHL1) was identified as abundant protein in rat and human beta cells, showing promising beta cell-selectivity, and was selected for further validation in standardized toxicity models. In vitro, H2O2-induced necrosis of INS-1 cells and human islets resulted in intracellular UCHL1 depletion and its extracellular discharge. In vivo, streptozotocin progressively depleted UCHL1 from islet cores and in 50% of animals, an associated plasma UCHL1 surge was detected preceding the GAD65 peak. UCHL1 was cleared with a half-life of 20min. Whole-body dynamic planar imaging of (99m)-Technetium-labeled UCHL1 indicated a rapid UCHL1 uptake in the liver and spleen, followed by urinary excretion of mainly proteolytic UCHL1 fragments. We conclude that LC-MS/MS proteomics is a useful tool to prioritize biomarkers for beta cell injury with promising molar abundance. Despite its consistent UCHL1 discharge by damaged beta cells in vitro, its in vivo use might be restrained by its rapid elimination from plasma.
  
  
  CONCLUSIONS
  Our bottom-up LC-MS/MS proteomics represents a pragmatic approach to identify protein-type biomarkers of pancreatic beta cell injury. UCHL1 successfully passed sequential validation steps of beta cell-selectivity, antigenicity and toxic discharge in vitro. Whole-body dynamic planar imaging of radiolabeled recombinant UCHL1 indicated rapid clearance through the liver, spleen and urinary excretion of proteolytic fragments, likely explaining non-consistent detection in vivo. Integration of kinetic biomarker clearance studies in the a priori selection criteria is recommended before engaging in resource-intensive custom development of sensitive immunoassays for clinical translation.
authors: Diabetes Research Center, Vrije Universiteit Brussel (VUB), Belgium; Department of Clinical Chemistry and Radio-immunology, Universitair Ziekenhuis Brussel, Belgium.
authors: B
authors: B
authors: Brackeva
authors: Diabetes Research Center, Vrije Universiteit Brussel (VUB), Belgium; Department of Clinical Chemistry and Radio-immunology, Universitair Ziekenhuis Brussel, Belgium.
authors: V
authors: V
authors: De Punt
authors: Department of Medical Biochemistry, Academisch Medisch Centrum, Amsterdam, Netherlands.
authors: G
authors: G
authors: Kramer
authors: Department of Clinical Chemistry and Radio-immunology, Universitair Ziekenhuis Brussel, Belgium.
authors: O
authors: O
authors: Costa
authors: Department of Clinical Chemistry and Radio-immunology, Universitair Ziekenhuis Brussel, Belgium.
authors: K
authors: K
authors: Verhaeghen
authors: Diabetes Research Center, Vrije Universiteit Brussel (VUB), Belgium.
authors: G
authors: G
authors: Stangé
authors: Department of Anatomopathology, Universitair Ziekenhuis Brussel, Belgium.
authors: J
authors: J
authors: Sadones
authors: In Vivo Cellular and Molecular Imaging Laboratory (ICMI), Vrije Universiteit Brussel (VUB), Belgium.
authors: C
authors: C
authors: Xavier
authors: Department of Medical Biochemistry, Academisch Medisch Centrum, Amsterdam, Netherlands.
authors: J M F G
authors: JM
authors: Aerts
authors: Department of Clinical Chemistry and Radio-immunology, Universitair Ziekenhuis Brussel, Belgium.
authors: F K
authors: FK
authors: Gorus
authors: Diabetes Research Center, Vrije Universiteit Brussel (VUB), Belgium; Department of Clinical Chemistry and Radio-immunology, Universitair Ziekenhuis Brussel, Belgium. Electronic address: geert.martens@uzbrussel.be.
authors: G A
authors: GA
authors: Martens
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D050417:Insulin-Secreting Cells / Q000378:metabolism* / Q000473:pathology; D009336:Necrosis / Q000378:metabolism / Q000473:pathology; D051381:Rats; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism*
chemical_list: D015415:Biomarkers; C482860:UCHL1 protein, human; C489034:UCHL1 protein, rat; D043222:Ubiquitin Thiolesterase
keywords: Biomarkers; Cell death; Insulin-secreting cells; Proteomics