Proteomics for protein expression profiling in neuroscience

Abstract

As the technology of proteomics moves from a theoretical approach to a practical reality, neuroscientists will have to determine the most appropriate applications for this technology. Neuroscientists will have to surmount difficulties particular to their research, such as limited sample amounts, heterogeneous cellular compositions in samples, and the fact that many proteins of interest are rare, hydrophobic proteins. This review examines protein isolation and protein fractionation and separation using two-dimensional electrophoresis (2-DE) and mass spectrometry proteomic methods. Methods for quantifying relative protein expression between samples (e.g., 2-DIGE, and ICAT) are also described. The coverage of the proteome, ability to detect membrane proteins, resource requirements, and quantitative reliability of different approaches is also discussed. Although there are many challenges in proteomic neuroscience, this field promises many rewards in the future.

Fig. 1

All proteomic experiments incorporate these experimental steps. Though most experiments occur in this order, some methods do proceed in a different order.

Fig. 2

Common methods for protein separation, quantitation, and identification.

Fig. 3

Difference in gel electrophoresis (DIGE) departs from standard 2-DE in that the samples are labeled with fluorescent dyes and mixed together before being run on the same IEF and second-dimension gel. A pool, created by mixing equal amounts of each sample analyzed, is run to both normalize quantitation across gels and for spiking in to increase the amount of protein to be picked for MS protein identification.

Fig. 4

Spot matching and quantitation with different two-dimensional electrophoresis approaches. (A) Spot matching is the process of determining which spot is the same across multiple gels. Two-dimensional DIGE aids matching by using an internal control channel present on each gel and fewer gels in total. (B) Quantitation of traditional 2-DE uses densitometry of matched spots. 2-DIGE improves quantitation by assessing abundance in relation to a normalization pool on each gel, thereby normalizing for gel to gel technical differences.

Fig. 5

Schematics of common ionization and mass analysis technologies (see text for details).