In-Depth Proteomic Analysis of the Porcine Retina by Use of a four Step Differential Extraction Bottom up LC MS Platform.

The eye of the house swine (Sus scrofa domestica Linnaeus, 1758) represents a promising model for the study of human eye diseases encircling neurodegenerative retina disorders that go along with proteomic changes. To provide an in-depth view into the "normal" (untreated & healthy) porcine retina proteome as an important reference, a proteomic strategy has been developed encircling stepwise/differential extraction, LC MS and peptide de novo sequencing. Accordingly, pooled porcine retina homogenates were processed by stepwise DDM, CHAPS, ASB14 and ACN/TFA extraction. Retinal proteins were fractionated by 1D-SDS PAGE and further analyzed by LC ESI MS following database and de novo sequencing related protein identification and functional analyses. In summary, >2000 retinal proteins (FDR < 1 %) could be identified by use of the highly reproducible and selective extraction procedure. Moreover, an identification surplus of 36 % comparing initial one step extraction to the four step method could be documented. Despite most proteins were identified in the DDM and CHAPS fraction, all extraction steps contributed exclusive proteins with nucleus proteins enriched in the final ACN/TFA fraction. Additionally, for the first time new non-annotated de novo peptides could be documented for the porcine retina. The generated porcine retina proteome reference map contributes importantly to the understanding of the pig eye proteome and the developed workflow has strong translational potential considering retina studies of various species.