Proteomic analysis of the mouse brain following protein enrichment by preparative electrophoresis

Abstract

Proteomics is a powerful technology to study the identity and levels of brain proteins. Changes of protein levels as well as modifications that occur in neurological disorders may be informative for the pathogenesis of these disorders and could result in the identification of potential drug targets and disease markers. To increase the capability of characterizing complex protein profiles, protein mixtures should be separated into simpler fractions, thus increasing the likelihood of detecting low-abundance proteins. Considering that low-abundance proteins are thought to be involved in important biological processes, identification of those low-copy-number gene products appears to be a scientific challenge. In the present study, proteomic analysis of adult mouse brain tissue was performed following enrichment by preparative electrophoresis. This was performed using the PrepCell apparatus in the presence of 0.1% lithium dodecyl sulfate. Samples were electrophoresed in a cylindrical polyacrylamide gel and the proteins of the fractions collected were first analyzed by 1-D and then by 2-DE. Protein identification was performed by MALDI-TOF-MS. The present analysis resulted in the identification of 360 different gene products. Among those were transport proteins, transcription activators, signal transduction molecules as well as proteins with a number of other functions. Preparative electrophoresis is an efficient method for the enrichment of proteins of low molecular mass and may be useful in the investigation of disorders of the central nervous system.

Rapid Label-Free Identification of Estrogen-Induced Differential Protein Expression In Vivo from Mouse Brain and Uterine Tissue

“…However, to put our results in perspectives, exploration of the rat brain proteome directly from whole brain tissue by 2D-GE had started with the identification of 210 proteins (a number comparable to what we covered by our rapid proteomics survey in the mouse cortex), which implicated the applicability of the methodology only to abundant brain proteins. Additional developments, including the use of large-gel formats to improve electrophoretic separation and attention to pre-electrophoretic enrichment of low-abundance gene products, , have maintained the viability of this methodology despite its recognized shortcomings upon analyses of complex protein mixtures , and, unfortunately, at the cost of decreasing throughput. The premise of the presented label-free approach to explore differential protein expressions should also be viewed in a similar context.…”

Current Status and Advances in Quantitative Proteomic Mass Spectrometry

“…Spectral counting is a protein-centric approach that is less reliable for trace and/or low mass proteins; and less responsive toward small changes in response (<2 orders of magnitude) [11, 55], favoring higher abundance “average” proteins [2], while lower identification rates for proteins with low sequence coverage and nontryptic or fewer peptides are a consequence of the methods used for identification as much as the dynamic range of the sample and the limited duty cycle of the MS instrument [56]. This approach has been modified into forms such as the exponentially modified protein abundance index [57] and absolute protein expression profiling [58]. …”