title: Quantitative Proteomic Analysis of Human Airway Cilia Identifies Previously Uncharacterized Proteins of High Abundance.
abstract: Cilia are essential to many diverse cellular processes. Although many major axonemal components have been identified and studied, how they interact to form a functional axoneme is not completely understood. To further our understanding of the protein composition of human airway cilia, we performed a semiquantitative analysis of ciliary axonemes using label-free LC/MSE, which identified over 400 proteins with high confidence. Tubulins were the most abundant proteins identified, with evidence of 20 different isoforms obtained. Twelve different isoforms of axonemal dynein heavy chain were also identified. Absolute quantification of the nontubulin components demonstrated a greater than 75-fold range of protein abundance (RSPH9;1850 fmol vs CCDC103;24 fmol), adding another level of complexity to axonemal structure. Of the identified proteins, ∼70% are known axonemal proteins. In addition, many previously uncharacterized proteins were identified. Unexpectedly, several of these, including ERICH3, C1orf87, and CCDC181, were present at high relative abundance in the cilia. RT-PCR analysis and immunoblotting confirmed cilia-specific expression for eight uncharacterized proteins, and fluorescence microscopy demonstrated unique axonemal localizations. These studies have provided the first quantitative analysis of the ciliary proteome and have identified and characterized several previously unknown proteins as major constituents of human airway cilia.
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University , Raleigh, North Carolina 27695, United States.
authors.forename: Kevin
authors.initials: K
authors.lastname: Blackburn
authors.affiliation: Marsico Lung Institute/UNC CF Research Center, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors.forename: Ximena
authors.initials: X
authors.lastname: Bustamante-Marin
authors.affiliation: Marsico Lung Institute/UNC CF Research Center, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors.forename: Weining
authors.initials: W
authors.lastname: Yin
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University , Raleigh, North Carolina 27695, United States.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Goshe
authors.affiliation: Marsico Lung Institute/UNC CF Research Center, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors.forename: Lawrence E
authors.identifier: 0000-0002-7558-6603
authors.initials: LE
authors.lastname: Ostrowski
mesh_terms: D054468:Axoneme / Q000235:genetics*; D002923:Cilia / Q000235:genetics*; D004398:Dyneins / Q000235:genetics / Q000302:isolation & purification; D005786:Gene Expression Regulation; D006801:Humans; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification; D020543:Proteome / Q000235:genetics*; D040901:Proteomics; D014404:Tubulin / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011506:Proteins; D020543:Proteome; D014404:Tubulin; D004398:Dyneins
keywords: airway; axoneme; cilia; dynein; mass spectrometry; proteomics; radial spoke; tubulin