Deep imaging: how much of the proteome does current top-down technology already resolve?
Effective proteome analyses are based on interplay between resolution and detection. It had been claimed that resolution was the main factor limiting the use of two-dimensional gel electrophoresis. Improved protein detection now indicates that this is unlikely to be the case. Using a highly refined protocol, the rat brain proteome was extracted, resolved, and detected. In order to overcome the stain saturation threshold, high abundance protein species were excised from the gel following standard imaging. Gels were then imaged again using longer exposure times, enabling detection of lower abundance, less intensely stained protein species. This resulted in a significant enhancement in the detection of resolved proteins, and a slightly modified digestion protocol enabled effective identification by standard mass spectrometric methods. The data indicate that the resolution required for comprehensive proteome analyses is already available, can assess multiple samples in parallel, and preserve critical information concerning post-translational modifications. Further optimization of staining and detection methods promises additional improvements to this economical, widely accessible and effective top-down approach to proteome analysis.
Molecular Physiology, and the UWS Molecular Medicine Research Group, School of Medicine, University of Western Sydney, Campbelltown, NSW, Australia.
Elise P
EP
Wright
Molecular Physiology, and the UWS Molecular Medicine Research Group, School of Medicine, University of Western Sydney, Campbelltown, NSW, Australia.
Kali A G
KA
Prasad
Proteomics Core Facility, Faculty of Science, University of Technology, Sydney, NSW, Australia.
Matthew P
MP
Padula
Molecular Physiology, and the UWS Molecular Medicine Research Group, School of Medicine, University of Western Sydney, Campbelltown, NSW, Australia.
Jens R
JR
Coorssen
D000818:Animals; D005260:Female; D008297:Male; D013058:Mass Spectrometry; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D051381:Rats
D020543:Proteome