2012- 21st Annual NC State Undergraduate Research Symposium

Session Time : 4/10/12 10:30 AM - 4/10/12 11:45 AM

Content Area : Biochemistry

Poster Appointment: NONE

Student Presenters :
Justyne Starr Hammond Biochemistry, Human Biology

Mentors and/or Co-Authors :
Michael Goshe Biochemistry

Abstract Title : Proteomic Analysis of the Human Respiratory Ciliary Membrane using Streptavidin Affinity Purification and Liquid Chromatography-Tandem Mass Spectrometry

Abstract :
Justyne Hammond, Lawrence Ostrowski, Kevin Blackburn, and Michael B. Goshe

Cilia are hair-like macromolecular organelles projecting from epithelial cell surfaces that line the respiratory tract. Through coordinated beating, cilia clear the airway of mucus and debris. Cilia are comprised of a conserved axonemal structure encased within the ciliary membrane. While the primary role of cilia is clearance of debris, it is possible that airway cilia have other roles including perception of mucus flow, bacteria, or viruses. These other sensory-type functions would likely be mediated through membrane associated proteins. Unfortunately, little is known regarding the protein composition of the ciliary membrane. While the ciliary membrane is likely to share common components with the epithelial cell membrane, it is also expected to contain unique proteins. To gain insight into the ciliary membrane composition, a proteomic analysis of the membrane was conducted. Following chemical biotinylation of surface membrane proteins in a airway epithelial cell culture, intact cilia were isolated, the ciliary membrane was solubilized with detergent, and the axoneme was removed by centrifugation. Biotinylated membrane proteins were bound to streptavidin beads and washed to remove nonspecifically bound proteins. Bead-bound membrane proteins were trypsinized in situ and analyzed by LC/MS/MS using an Orbitrap Elite mass spectrometer. A number of ciliary membrane proteins were identified, including annexins A1 and A3, putative annexin A2-like protein, brain acid soluble protein 1, Ras GTPase activating-like protein, sodium-dependent phosphate transport protein 2B, transgelin-2, and potassium transporting ATPase a-chain B.