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Reference: Tran JC, et al. (2009) Evaluation of a solution isoelectric focusing protocol as an alternative to ion exchange chromatography for charge-based proteome prefractionation. J Chromatogr B Analyt Technol Biomed Life Sci 877(8-9):807-13

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Abstract


Solution isoelectric focusing (sIEF) is evaluated relative to ion exchange chromatography (IEC) as a preferred charge-based prefractionation tool for proteome mixtures. While IEC is extensively employed for proteome prefractionation prior to MS analysis, we demonstrate here that conventional salt gradient IEC has significant shortcomings compared to sIEF. Here, we critically evaluated a custom eight-channel sIEF device for intact protein separation, relative to strong cation exchange (SCX) and strong anion exchange (SAX) chromatography. The resolution, recovery, and uniformity of separation obtained with our sIEF device were comparable or superior to that of optimized IEC separations. Most importantly for intact proteins, sIEF separations strongly correlate with the proteins' isoelectric point, which contrasts with IEC where no correlation was observed. To validate the sIEF platform for proteome analysis, prefractionation through sIEF resulted in the confident identification of a greater number of proteins from yeast (211) following LC-MS/MS, relative to those obtained through SAX (173) or SCX (148).

Reference Type
Journal Article
Authors
Tran JC, Wall MJ, Doucette AA
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