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Attomole protein analysis by CIEF with LIF detection

2009; Wiley; Volume: 30; Issue: 2 Linguagem: Inglês

10.1002/elps.200800498

1522-2683

Lauren M. Ramsay, Jane A. Dickerson, Norman J. Dovic̀hi,

Mass Spectrometry Techniques and Applications

Abstract 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 ε ‐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 p I . 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 β ‐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.