Protein Microarrays and Protein Microarrays and detection via MALDI-TOF:detection via MALDI-TOF:

Project OverviewProject Overview

Kyle NordquistKyle Nordquist

Advisor: Dr. Andrew LinkAdvisor: Dr. Andrew Link

Microarray FoundationMicroarray Foundation

DNA microarrays DNA microarrays method of high-method of high-throughput gene throughput gene analysisanalysis

DNA nucleotides with DNA nucleotides with specific gene specific gene expression spottedexpression spotted

““Chip” probed Chip” probed fluorescentlyfluorescently

Research PurposesResearch Purposes

Different Different InteractionsInteractions DNA-DNADNA-DNA DNA-small DNA-small

moleculemolecule DNA-antibodyDNA-antibody

Protein ApplicationsProtein Applications

Interactions at Protein Interactions at Protein levellevel

Protein-Drug Protein-Drug relationshipsrelationships

Crucial Crucial pharmaceutical toolpharmaceutical tool

ComplicationsComplications

High-throughput purification --tediousHigh-throughput purification --tedious

Proteins must remain in native Proteins must remain in native conformation to be of any useconformation to be of any use

Binding affinitiesBinding affinities Tagging (His/GST)Tagging (His/GST)

GST-purificationGST-purification

Yeast genome library Yeast genome library obtained in 96-well obtained in 96-well formatformat

Cells must be grown Cells must be grown and lysedand lysed

Overexpressed Overexpressed proteins must be proteins must be extractedextracted

ContaminationContamination

SpottingSpotting

Vanderbilt Microarray Vanderbilt Microarray Shared Resource Shared Resource CenterCenter

Numerous surface Numerous surface chemistries to be chemistries to be testedtested

Current DetectionCurrent Detection

Published literature using fluorescent Published literature using fluorescent detection datadetection data

Shows protein intensityShows protein intensity

Fails to show:Fails to show: Size of proteinsSize of proteins Interaction affinityInteraction affinity

MALDI-TOF MSMALDI-TOF MS

Matrix-Assisted Laser Matrix-Assisted Laser Desorption and Desorption and Ionization Time-of-Ionization Time-of-Flight Mass Flight Mass SpectrometrySpectrometry

Has not been used to Has not been used to quantify protein chip quantify protein chip datadata

Possible Advantages of using a Possible Advantages of using a MALDI detection systemMALDI detection system

Specific mass data in reference to binding Specific mass data in reference to binding interactionsinteractions Classify what molecules bound to what Classify what molecules bound to what

proteinsproteins

Easy to distinguish unknown proteins by Easy to distinguish unknown proteins by weightweight

Less time for preparationLess time for preparation