Quantitative Proteomic Analysis of Human Airway Cilia Identifies Previously Uncharacterized Proteins of High Abundance

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.