Attomole protein analysis by CIEF with LIF detection.
We have coupled CIEF with an LIF detector that is based on a post-column sheath flow cuvette. We employed Chromeo P503 as a fluorogenic reagent to label proteins before analysis. This reagent reacts with the epsilon-amine of lysine residues, preserving the cationic nature of the residue; labeled proteins generate extremely sharp peaks in CIEF. A set of four standard proteins generated a linear relationship between migration time and pI. A protein homogenate prepared from a Barrett's esophagus cell line resolved over 100 components in a 40 min separation. Detection limits for Chromeo P503-labeled beta-lactoglobulin were 5 amol injected into the capillary. Fluorescent impurities present in the ampholytes generated a large background signal that degraded the detection limit by four orders of magnitude compared with other forms of capillary electrophoresis with this detector.
Ramsay
Lauren M
LM
Department of Chemistry, University of Washington, Seattle, 98195-1700, USA.
Dickerson
Jane A
JA
Dovichi
Norman J
NJ
D002138:Calibration; D002460:Cell Line; D002477:Cells / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D011506:Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors
D005456:Fluorescent Dyes; D011506:Proteins