High-Throughput Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry Method for Quantification of Hepcidin in Human Urine

Levels of the peptide hormone hepcidin negatively correlate with systemic iron status and are increased in disorders in which iron metabolism is secondarily disregulated, such as the anemia of chronic disease. Consequently, the ability to measure hepcidin in the clinical setting may have diagnostic value for a broad range of indications. We describe a novel quantitative matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry assay for hepcidin in human urine which involves (i) direct enrichment from minute volumes (5 μL) of minimally treated urine on the surface of a functionalized chip, (ii) quantification by the use of a stable isotope labeled internal standard, and (iii) analysis by MALDI-TOF. Performance features include a wide linear range (1−1000 nM; LOQ 2.5 nM), high accuracy (90−110% recovery) and precision (intraday CV 12.11%; interday CV 13.21%), and a strong correlation upon interlaboratory cross validation with an existing immunoassay. The assay is simple, accurate, and efficient, and the high-throughput performance features of the assay make large-scale clinical research studies feasible

High-Throughput Assay to Screen Small Molecules for Their Ability to Prevent Sickling of Red Blood Cells

Sickle cell disease (SCD) is an inherited disorder of hemoglobin (Hb); approximately 300,000 babies are born worldwide with SCD each year. In SCD, fibers of polymerized sickle Hb (HbS) form in red blood cells (RBCs), which cause RBCs to develop their characteristic “sickled” shape, resulting in hemolytic anemia and numerous vascular complications including vaso-occlusive crises. The development of novel antisickling compounds will provide new therapeutic options for patients with SCD. We developed a high-throughput “sickling assay” that is based on an automated high-content imaging system to quantify the effects of hypoxia on the shape and size of RBCs from HbSS SCD patients (SS RBCs). We used this assay to screen thousands of compounds for their ability to inhibit sickling. In the assay, voxelotor (an FDA-approved medication used to treat SCD) prevented sickling with a z′-factor > 0.4, suggesting that the assay is capable of identifying compounds that inhibit sickling. We screened the Broad Repurposing Library of 5393 compounds for their ability to prevent sickling in 4% oxygen/96% nitrogen. We identified two compounds, SNS-314 mesylate and voxelotor itself, that successfully prevented sickling. SNS-314 mesylate prevented sickling in the absence of oxygen, while voxelotor did not, suggesting that SNS-314 mesylate acts by a mechanism that is different from that of voxelotor. The sickling assay described in this study will permit the identification of additional, novel antisickling compounds, which will potentially expand the therapeutic options for SCD