Authors: Javad Nazarian; Yetrib Hathout; Eric P Hoffman; Bindesh Shrestha; Akos Vertes

Using proteomic analyses of the electroplaque tissue to understand the complex system of mammalian neuromuscular junction.

The neuromuscular junction (NMJ) has been used as a model synapse, where nerve cells communicate with and activate muscle cells. Most effort has been dedicated to understanding electrical transmission and signal transduction, and the signals from the nerve that initiate and maintain the NMJ cellular specialization at the point of nerve/muscle contact. However little is known about the protein makeup of the NMJ. The Torpedo electroplaque, which is primarily comprised of NMJs, has been extensively used as a model to understand the electrical, biophysical, and molecular properties of the NMJ in vertebrates. In this study we used the Torpedo californica electroplaque to explore the proteome of the NMJ.

Frozen torpedo californica electroplaque (150mg) was grinded in liquid nitrogen. Digitonin containing buffer was used to extract soluble proteins, while the remaining membrane fraction was treated with high pH (11) Na2CO3 to extract integral membrane proteins. The cytosolic fraction (250ug) was then processed for 2-DG (17cm pH 3-11) and MALDI-TOF-TOF analysis of in-gel digested spots. The membrane fraction (200ug) was digested with trypsin and/or proteinase K. Resulting peptides were analyzed by LC/MS and MS/MS using nano-LC-packing system coupled to a Thermo Finnigan LTQ instrument. The proteins were identified using Sequest and Mascot search engines.

In this study, we have generated a partial proteome map of Torpedo electroplaque. Overall, the gel array contained more than 300 spots from which 85 were confidently identified using MALDI-TOF-TOF MS/MS and database search against NCBInr database. All of these proteins have human or mice homologues. However, several proteins with distinct mass fingerprints did not match any of the known proteins in the database. These proteins were manually matched using Blast MS of short sequence tags. Additionally, Torpedo electroplaque was found to contain 12 isoforms for creatine kinase with different pI and molecular masses. Sequence homology between these isoforms was estimated to be around 40% as judged from de novo sequencing or the peptides from these isoforms. Analysis of the membrane fraction resulted in the identification of 140 proteins which were identified by database mining of homologous sequences in human and mice. Overall, the survey of the proteome of Torpedo californica, for which a genomic database does not currently exists, resulted in identification of a total of 225 unique proteins identified by trans-species homology search. A handful of known NMJ-associated proteins are within the obtained list. Defining molecular players associated with the torpedo electroplaque may bring insight into proteins involved in the NMJs which are difficult to obtain due the relatively small size of NMJ in human or mice (30nm). Further functional characterization of these genes/proteins may expand our understanding of the factors vital to NMJ stability and function.