Determination of picomolar concentrations of proteins using novel amino reactive chameleon labels and capillary electrophoresis laser-induced fluorescence detection.
Py-1 and Py-6 are novel amino-reactive fluorescent reagents. The names given to them reflect that they consist of a pyrylium group attached to small aromatic moieties. Upon reaction with a primary amine there is a large spectral shift in the reagent, rendering them effectively fluorogenic. In this study, these reagents were used to label a test protein, (human serum albumin), and the sample was analyzed by capillary electrophoresis and laser-induced fluorescence detection. Detection limits after a 60 min labeling reaction at 22 degrees C (Py-1) and 50 degrees C (Py-6) were 6.5 ng/mL (98 pM) for Py-1 and 1.2 ng/mL (18 pM) for Py-6. Separation of immunoglobulin G (IgG), human serum albumin, lipase, and myoglobin after labeling with Py-6 were performed. The method was further modified to make it amenable to automation. Unlike many other amino reactive reagents used to label protein amino groups, reaction with Py-1 and Py-6 do not alter the charge of the protein and the advantage of this with respect to electrophoretic separations is discussed.
Craig
Douglas B
DB
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Regensburg, Germany. d.craig@uwinnipeg.ca
Wetzl
Bianca K
BK
Duerkop
Axel
A
Wolfbeis
Otto S
OS
D000588:Amines; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification; D007834:Lasers; D008049:Lipase / Q000302:isolation & purification; D009211:Myoglobin / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis*; D012709:Serum Albumin / Q000302:isolation & purification
D000588:Amines; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D009211:Myoglobin; D011506:Proteins; D012709:Serum Albumin; D008049:Lipase