Stacking and separation of protein derivatives of naphthalene-2,3-dicarboxaldehyde by CE with light-emitting diode induced fluorescence detection.
We describe the stacking and separation of proteins by CE under discontinuous conditions in conjunction with light-emitting diode induced fluorescence (LEDIF) detection using a violet LED at 405 nm. The proteins were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) to form NDA-protein derivatives prior to CE-LEDIF analysis. During the separation, poly(ethylene oxide) (PEO) solution containing CTAB enters from the cathodic inlet to the capillary via electroosomotic flow (EOF). The optimum conditions are: the capillary was filled with 50 mM glycine buffer (pH 9.0) containing 1.0 mM CTAB, NDA-protein derivatives were prepared in deionized water containing 1.0 mM CTAB, and 0.6% PEO was prepared in 50 mM glycine (pH 9.0) containing 2.0 mM CTAB. The analysis of four NDA-protein derivatives is fast (<3 min), with RSD <1.5% in terms of migration time. In order to improve the sensitivity of NDA-protein derivatives, a stacking approach based on increases in viscosity and electric field, as well as sieving was applied. The efficient stacking approach provides LODs (S/N = 3) of 2.41, 0.59, 0.61, and 4.22 nM for trypsin inhibitor, HSA, beta-lactoglobulin, and lysozyme, respectively. In addition, we also applied the stacking approach to determination of the concentration of HSA in one urine sample, which was determined to be 0.31 +/- 0.05 microM (n = 3).
Department of Chemistry, National Taiwan University, Taipei, Taiwan.
Tai-Chia
TC
Chiu
Wu-Chun
WC
Tu
Huan-Tsung
HT
Chang
D000419:Albuminuria / Q000652:urine; D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D053841:Electroosmosis / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D007782:Lactoglobulins / Q000302:isolation & purification; D009113:Muramidase / Q000302:isolation & purification; D009281:Naphthalenes / Q000302:isolation & purification*; D009862:Online Systems; D011092:Polyethylene Glycols; D011506:Proteins / Q000302:isolation & purification*; D012709:Serum Albumin / Q000302:isolation & purification; D014361:Trypsin Inhibitors / Q000302:isolation & purification
D002593:Cetrimonium Compounds; D007782:Lactoglobulins; D009281:Naphthalenes; D011506:Proteins; D012709:Serum Albumin; D014361:Trypsin Inhibitors; D011092:Polyethylene Glycols; C027717:2,3-naphthalenedicarboxaldehyde; D009113:Muramidase; D000077286:Cetrimonium