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J Biomol Tech. 2010 September; 21(3 Suppl): S56–S57.
PMCID: PMC2918181

Improved Coverage of the Proteome using Gel-Eluted Liquid Fraction Entrapment Electrophoresis and Mass Spectrometry

I. Isaac, C. Brown, and C. Witkowski
Protein Discovery, Inc., Knoxville, TN, United States



Due to limitations of LC/MS/MS, orthogonal separation methods for reducing sample complexity are needed. Recently, the technique of Gel-Eluted Liquid Fraction Entrapment Electrophoresis (GELFREE) was introduced (Anal Chem, 80, 1568-1573). Using this approach, one can perform a preparative scale SDS PAGE separation to fractionate the sample according to protein molecular weight. What makes this approach unique is that the proteins are eluted from the gel matrix and collected in liquid fractions. The advantages of the gel-based separation for intact proteins are realized (e.g. high loading capacity and high resolution) with near quantitative recovery (>95%). This presentation will demonstrate the use of this technique along with mass spectrometry. This strategy increases the proteome coverage and the addition of intact molecular weight information increases confidence in identification.All experiments were performed using the Gelfree™ 8100 Fractionation System and pre-cast gel cartridges provided (Protein Discovery, Inc.). The performance characteristics were evaluated using S. Cerevisiae and bovine liver lysates fractionated using Gelfree 8100 into 12 fractions, ranging from 3.5-150 kDa. The fractions were treated to remove SDS, reduced, alkylated, and digested with trypsin. The digests were analyzed using nano-LC/MS/MS on an ion trap mass spectrometer. Control samples were prepared without fractionation. Fractionation using GELFREE increases the number of confident protein identifications by a factor of 3-5 fold. In one example, preliminary results increased the number of unique proteins identified in a single experiment from 250 proteins to 751 proteins in the case of S. Cerevisiae. Additionally, due to the molecular weight-based separation provided by GELFREE, proteins having multiple forms of differing molecular weight can be readily distinguished. Method for the use of Gelfree 8100 for both bottom-up and top-down experiments will be presented.

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