Array based capillary IEF with a whole column image of laser-induced fluorescence in coupling to capillary RPLC as a comprehensive 2-D separation system for proteome analysis.
Based on array CIEF (ACIEF) and a novel whole column imaging detection (WCID), a comprehensive 2-D system with laser-induced fluorescence was developed for protein mapping. By coupling capillary RPLC (CRPLC) as the first dimension and ACIEF as the second dimension, a high-throughput and high-resolution proteomic expression profiling was obtained. An array of up to 60 capillaries was assembled, with electrical connections made through filling small breaks, created on each capillary at positions of buffer reservoirs, with a porous polymer. A whole column image system with laser-induced fluorescence (LIF) was devised. Spot excitation was performed with a laser converted to produce linear light, and a CCD camera was employed to take images of the protein fluorescence during line laser scanning of the capillary array. Quantitative detection of thousands of focusing protein bands in the capillary array was achieved. Details on the capillary array fabrication and scanning LIF detection system devices are discussed. The efficiency of this CRPLC-ACIEF-LIF-WCID system was further demonstrated using samples of soluble proteins extracted from liver cancer tissue. The overall peak capacity was estimated to be around 18 000 in an analysis time of less than 3 h. The reproducibility of consecutive runs and different columns were assessed as having an RSD of 1.5% and 2.2% in focusing positions, respectively.
Mao
Yu
Y
Department of Chemistry and Research Center for Proteome, Fudan University, Shanghai, PR China.
Li
Yan
Y
Zhang
Xiangmin
X
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002853:Chromatography, Liquid; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes; D007525:Isoelectric Focusing; D007834:Lasers; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D051379:Mice; D010449:Peptide Mapping; D040901:Proteomics; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism
D005456:Fluorescent Dyes; D014357:Trypsin