In-depth Proteomic Analysis of Mouse Cochlear Sensory Epithelium by Mass Spectrometry

Proteomic analysis of sensory organs
such as the cochlea is challenging
due to its small size and difficulties with membrane protein isolation.
Mass spectrometry in conjunction with separation methods can provide
a more comprehensive proteome, because of the ability to enrich protein
samples, detect hydrophobic proteins, and identify low abundant proteins
by reducing the proteome dynamic range. GELFrEE as well as different
separation and digestion techniques were combined with FASP and nanoLC–MS/MS
to obtain an in-depth proteome analysis of cochlear sensory epithelium
from 30-day-old mice. Digestion with LysC/trypsin followed by SCX
fractionation and multiple nanoLC–MS/MS analyses identified
3773 proteins with a 1% FDR. Of these, 694 protein IDs were in the
plasmalemma. Protein IDs obtained by combining outcomes from GELFrEE/LysC/trypsin
with GELFrEE/trypsin/trypsin generated 2779 proteins, of which 606
additional proteins were identified using the GELFrEE/LysC/trypsin
approach. Combining results from the different techniques resulted
in a total of 4620 IDs, including a number of previously unreported
proteins. GO analyses showed high expression of binding and catalytic
proteins as well as proteins associated with metabolism. The results
show that the application of multiple techniques is needed to provide
an exhaustive proteome of the cochlear sensory epithelium that includes
many membrane proteins. The mass spectrometry proteomics data have
been deposited to the ProteomeXchange Consortium with the data set
identifier PXD000231.