Development of Multiple Reaction Monitoring Assay for Induced Pluripotent Cells
Clarkson, Trent M.
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It was posited that cells can be quantitated based on the unique proteins they produce. These proteins are in low abundance and their numbers relate directly to the cells that produce them. Multiple reaction method, a relatively new technique used in tandem mass spectrometry, demonstrated the ability to quantitate cells in this manner. In this project, an attempt was made to find the limits of detection using multiple reaction mechanism as a means of evaluating whether this can be a viable method for detecting induced pluripotent stem (iPS) cells. A developed protocol using MRM would be later employed by Dr. Gan Wang of Wayne State University to assess the efficiency of a protocol he has developed to induce pluripotency in somatic cells thus creating iPS cells. To increase the limit of detection using a multiple reaction method, solid phase extraction and SDS-PAGE protocols were used to purify proteins and separate them from one another. At the time of the writing of this report, this project has not yet been finished; however, there has been considerable progress made. A solid phase extraction protocol has been developed that achieved in some cases the orthogonal separation that would allow for a lower limit of detection. Using fluorescein as a technique for mapping SDS-PAGE gels was concluded to be too troublesome and inefficient for the purposes of this project. Furthermore, spectra of the ions produced by proteins unique to iPS cells looked fairly promising in that they had high signals and suggested the multiple reaction mechanism could be used as a means of quantitating iPS cells.