Automated Liquid Chromatography-Mass Spectrometry-Based Insulin Detection

Alton, Dominick J., Danita M. Dreffs, Braydon L. Dymm, Melanie M. Smalley, Sabrina I. Wadood, Tatum Y. Zurawski, and Kendra R. Evans

Biological molecules throughout the body respond to both external and internal stimuli in a dynamic manner.  For example, recent evidence suggests long-term (2-4 h or more) rhythms or cycles exist in the secretion and uptake of the endocrine hormone, insulin.  These rhythms are not well-understood but are of particular interest because biological cycles are believed to have an important role in regulating blood glucose, and changes in the rhythms appear to be associated with metabolic conditions such as diabetes and obesity.  To enable closer study of these rhythms, it is important to develop reliable, rapid methods of monitoring changes in insulin released by cells. Typically, hormone levels are monitored via radioimmunoassays or enzyme-linked immunosorbent assays; both assays are sensitive but are labor- and time-intensive and costly, and the assays cannot be used to detect multiple hormones simultaneously.  We have developed a liquid chromatography-mass spectrometry-based method for automated sampling, injection, and on-line detection of multiple proteins simultaneously.  The system employs a 10-port switching valve for computer-controlled injections and can be coupled to endocrine cells for continuous injection and detection of hormones.  The multi-analyte detection capabilities of the system have been characterized using model protein standards.  The method allows continuous insulin monitoring for 24 h or longer with 22-min temporal resolution.  Additionally, a liquid chromatograph-mass spectrometry analysis of insulin release from INS-1 cells was performed to demonstrate the capabilities of the instrumentation to detect insulin release from living cells in culture media.