Quantitative proteomics of rat and human pancreatic beta cells.

Data set description: This data set is composed by label-free alternate-scanning LC-MS/MS proteomics analysis human and Wistar rat pancreatic islet endocrine cells. The mass spectrometry data of the human and rat pancreatic beta cells and the resulting proteome search output from ProteinLynx GlobalSERVER (PLGS) have been deposited to the ProteomeXchange Consortium [1] via the PRIDE partner repository with the dataset identifiers PXD001539 (human) and PXD001816 (rat). From these mass spectrometry data, 'relative molar amount units' between cell types and across species were calculated. Biological relevance: These data provide a quantitative view on the unfractionated proteomes of human and rat beta and alpha cells. It is likely biased towards the proteins with higher molar abundance, relating to core functional pathways, but also includes several proteins with an islet-enriched expression. The quality of the cell preps is state-of-the-art, and the label-free quantitation is both precise and accurate, allowing detailed quantitative analysis.

Potential of UCHL1 as biomarker for destruction of pancreatic beta cells.

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. BIOLOGICAL SIGNIFICANCE: 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.

LC-MS/MS identification of doublecortin as abundant beta cell-selective protein discharged by damaged beta cells in vitro.

There is a clinical need for plasma tests that can directly detect injury to pancreatic beta cells in type 1 diabetes. Such tests require biomarkers that are abundantly and selectively released into plasma by damaged beta cells. We combined LC-MS/MS proteomics and tissue-comparative transcriptomics of FACS-purified beta cells for bottom-up identification of candidate markers. Less than 10% of 467 proteins detected in beta cells showed endocrine-enriched expression. One surprising candidate was the neuronal migration marker doublecortin: in situ analysis revealed uniform doublecortin expression in the cytoplasm of all beta cells. Western blotting and real-time PCR confirmed its strong beta cell-selectivity outside the brain and its high molar abundance, indicating promising biomarker properties in comparison to GAD65, a more established marker of beta cell injury. DCX potential was validated in vitro: chemically-induced necrosis of rat and human beta cells led to a discharge of intracellular doublecortin into the extracellular space, proportionate to the amount of injured cells, and similar to GAD65. In vivo, recombinant DCX showed favorable pharmacokinetic properties, with a half-life in plasma of around 3h. Combined, our findings provide first proof-of-principle for doublecortin as biomarker for beta cell injury in vitro, advocating its further validation as biomarker in vivo.