Towards online reduction and protein digestion of secreted proteins from pancreatic islets of Langerhans related to diabetes mellitus type I

What is diabetes mellitus type 1, and how can liquid chromatography assist in the search for a cure? The human pancreas is involved in control of metabolism and energy consumption by both an exocrine and an endocrine function. In the exocrine tissue, a multitude of digestive enzymes are produced and secreted into the small intestine to assist in breaking down proteins, fats and carbohydrates. The endocrine tissue, islets of Langerhans (5% of the total pancreatic mass) are responsible for regulation of the blood glucose levels. The vital metabolic hormone insulin is synthesized in one of five major cell types in the islets, called β-cells [1]. However, there are two types of chronic diseases related to insulin deficiency; an autoimmune disease causes selectively destruction of the β-cells in the islets or the body cannot effectively use the produced insulin, called diabetes mellitus type 1 and type 2, respectively. Diabetes mellitus type 1 or type 2 have been diagnosed in 250 000 Norwegians (approx. 4.7% of the Norwegian population), where 28 000 have been diagnosed with type 1 diabetes mellitus (0.5%) [2]. The most common treatment is exogenous insulin therapy. When insulin was discovered in 1920s the mortality of diabetes type 1 was reduced from a guaranteed fatal diagnosis to a manageable condition with a normal lifespan within the 1950s. However, secondary complications due to diabetic nephropathy, retinopathy and neuropathy led to a renewed interest in β-cell replacement therapy. Progress in β-cell replacement with either pancreas or islet transplantation has led to impressive 1- and 5-year insulin independence rates of approximately 85% and 50 %, respectively [3]. To assist in further progress in β-cell replacement therapy, liquid chromatography with mass spectrometry detection (LC-MS) of the secreted proteins from the pancreas and the islets will aid in the understanding of the treatment of diabetes mellitus type 1. In this work, we seek to enable fully online determination of proteins (in a bottom-up approach) by combining reduction and protein digestion upstream LC-MS (Figure 1). Online protein digestion has been successfully executed with immobilized enzyme reactors (IMERs) in different formats [4-6], but most of the samples have been reduced and alkylated prior to online digestion. We hope to enable real-time determination of proteins with disulfide bonds (such as insulin) by incorporation of a photochemical system for rapid reduction of disulfide bonds [7].