Proteome Analysis of Human Plasma with the ProteomeLab™ PF 2D System
Michael H. Simonian, Ph.D., and Edna Betgovargez
Beckman Coulter, Inc.
The sample is injected onto the 1st dimension chromatofocusing column, where proteins are separated by their isoelectric point (pI) with a pH gradient generated on the column. It is recommended that 1 to 5 mg of protein be injected in order to detect low abundance proteins. The combination fraction collector and auto-injector, FC/I Module, collects fractions from the 1st dimension based on pH. The fractions are collected onto a 96-deepwell plate. When the 1st dimension is complete, the FC/I Module becomes the auto-injector for the 2nd dimension. Here, the 1st dimension fractions are separated by reversed phase chromatography and the proteins are detected by UV absorbance. With this system, the 1st and 2nd dimensions occur sequentially in an automatic manner.
1st Dimension Separation, Chromatofocusing
Materials
The chemistry components consist of the HPCF chromatofocusing column and four solvents, Start Buffer (pH 8.5), Eluent Buffer (pH 4.0), 1 M NaCl, and water.
Method
The 1st-dimension separation was done at ambient temperature with a flow rate of 0.2 mL/min, and absorbance of the column effluent was monitored at 280 nm. Using the Direct Control mode of the software, the column was first equilibrated with 30 volumes (130 minutes) of Start Buffer. The method was then started with the injection of 5 mg of protein sample. Twenty minutes after the sample was injected and the 280 nm absorbance baseline was achieved, the pH gradient was generated by starting the Eluent Buffer, which was done by the programmed switching of the solvent selector valve in the HPCF Module. When the effluent reached pH 4.00 at 115 minutes after the injection of sample, the column was washed with 10 volumes of 1 M NaCl (45 minutes) followed by 10 volumes of water (45 minutes). These washes were programmed to take effect with the switching of the HPCF Module's solvent selector valve. During the pH gradient portion of the run, fractions at 0.3-pH intervals were collected as detected by the pH monitor, which controlled the fraction collection by the FC/I Module. During other portions of the run, fractions were collected by time at 5 min/fraction.
2nd Dimension Separation, Reversed Phase
Materials
The HPRP reversed-phase column was used with 0.1% TFA in water (Solvent A) and 0.08% TFA in acetonitrile (Solvent B).
Method
The second dimension separation was done at 50º C with a flow rate of 0.75 mL/min and absorbance of the column effluent was measured at 214 nm. The column was first equilibrated with 10 volumes (8 minutes) of 100% Solvent A prior to each injection. From each 1st dimension fraction, 200 µL were injected and, two minutes after injection, the column was eluted with a gradient of 0-100% Solvent B over 30 minutes. At the conclusion of this gradient, 100% Solvent B was maintained for 5 volumes (4 minutes) prior to re-equilibration to 100% Solvent A.
* All trademarks are the property of their respective owners. Where applicable, the PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman-LaRoche, Ltd.
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