Capillary sieving electrophoresis and micellar electrokinetic capillary chromatography produce highly correlated separation of tryptic digests

✍ Scribed by Jane A. Dickerson; Norman J. Dovichi

• Publisher: John Wiley and Sons
• Year: 2010
• Tongue: English
• Weight: 165 KB
• Volume: 31
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/elps.201000200

✦ Synopsis

Abstract

We perform 2‐D capillary electrophoresis on fluorescently labeled proteins and peptides. Capillary sieving electrophoresis (CSE) was performed in the first dimension and MEKC was performed in the second. A cellular homogenate was labeled with the fluorogenic reagent FQ and separated using the system. This homogenate generated a pair of ridges; the first had essentially constant migration time in the CSE dimension, while the second had essentially constant migration time in the MEKC dimension. In addition, a few spots were scattered through the electropherogram. The same homogenate was digested using trypsin, and then labeled and subjected to the 2‐D separation. In this case, the two ridges observed from the original 2‐D separation disappeared and were replaced by a set of spots that fell along the diagonal. Those spots were identified using a local‐maximum algorithm and each was fit using a 2‐D Gaussian surface by an unsupervised nonlinear least squares regression algorithm. The migration times of the tryptic digest components were highly correlated (r=0.862). When the slowest migrating components were eliminated from the analysis, the correlation coefficient improved to r=0.956.