Protein Microarrays and detection via MALDI-TOF: Project Overview Kyle Nordquist Advisor: Dr. Andrew Link

Microarray Foundation DNA microarrays method of high- throughput gene analysis DNA nucleotides with specific gene expression spotted “Chip” probed fluorescently

Research Purposes Different Interactions DNA-DNA DNA-small molecule DNA-antibody

Protein Applications Interactions at Protein level Protein-Drug relationships Crucial pharmaceutical tool

Complications High-throughput purification --tedious Proteins must remain in native conformation to be of any use Binding affinities Tagging (His/GST) Tagging (His/GST)

GST-purification Yeast genome library obtained in 96-well format Cells must be grown and lysed Overexpressed proteins must be extracted Contamination

Spotting Vanderbilt Microarray Shared Resource Center Numerous surface chemistries to be tested

Current Detection Published literature using fluorescent detection data Shows protein intensity Fails to show: Size of proteins Size of proteins Interaction affinity Interaction affinity

MALDI-TOF MS Matrix-Assisted Laser Desorption and Ionization Time-of- Flight Mass Spectrometry Has not been used to quantify protein chip data

Possible Advantages of using a MALDI detection system Specific mass data in reference to binding interactions Classify what molecules bound to what proteins Classify what molecules bound to what proteins Easy to distinguish unknown proteins by weight Less time for preparation