271993 Evaluation of Stain-Free Chemistry for Gel Electrophoresis Based Shotgun Proteomic Workflows

Tuesday, October 30, 2012: 10:15 AM
Fayette (Westin )
Sricharan Bandhakavi1, Tim Wehr1, Todd Markowski2, LeeAnn Higgins2, Xiaoyi Xu1, Aran Paulus1 and Christopher Belisle1, (1)Bio-Rad Laboratories, Hercules, CA, (2)University of Minnesota, Minneapolis, MN

In processing their samples for shotgun proteomics, researchers commonly use SDS-PAGE/gel electrophoresis to initially resolve complex biological samples.  Subsequently, the gel is stained for visualization of protein lane(s), and sliced into discrete ‘fractions’ along each lane.  Finally, each fraction is processed for in-gel protein digestion, peptide purification, and LC-MS/MS analysis. This workflow, in its entirety, is commonly referred to as ‘GeLC-MS’ and widely used for identifying resident proteins within complex samples of interest.

Several reports have demonstrated that compared to 'in-solution' protein digestion and fractionation, GeLC-MS enables higher total protein identifications with particular benefits for membrane/hydrophobic proteins (Fang et al., 2010 J. Proteome Res., Piersma et al. 2010 J. Proteome Res.).  In spite of these inherent benefits, improvements in the traditional GeLC-MS workflow (eg: in terms of speed and ease of processing) are needed to widen its adoption among researchers.  Here, we investigated the suitability of tris-glycine extended-life formulation (TGX) gels with 'stain-free chemistry' (a photochemical reaction ‘in-gel’ allowing for proteins to be visualized by fluorescence) for faster and simplified GeLC-MS analysis of a complex biological sample. 

Identical amounts of mouse 3T3 whole cell lysate were resolved using traditional Tris-HCl (4-15%) versus TGX stain-free (4-15%) gels and processed via GeLC-MS.  Compared to Tris-HCl gels, TGX stain-free gels 'ran' faster allowing electrophoresis to be completed in half the time.  For subsequent visualization of protein lanes, Coomassie staining/destaining was used for Tris-HCl gels, whereas a simple 30 second UV transillumination step was sufficient for TGX stain-free gels.  Fractions were collected across each gel lane and proteins identified via analysis of purified, fractionated tryptic peptides on a Velos-Orbitrap mass spectrometer.

Nearly identical numbers of proteins, peptides, and spectra were identified at identical spectral identification rates using either Tris-HCl or TGX stain-free gels.  Thus, incorporating TGX stain-free gels represents a valid approach for simplifying current GeLC-MS worflows used in shotgun proteomic studies. Details of our study and a simplified GeLC-MS workflow using TGX stain-free gels will be presented.


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