Development of a two-dimensional protein-peptide separation protocol for comprehensive proteome measurements.
We have developed an effective two-dimensional fractionation protocol of complex proteome mixtures that extends the ability to conduct more comprehensive proteome measurements. A sample containing intact proteins extracted from Saccharomyces cerevisiae was fractionated by liquid phase isoelectric focusing, followed by tryptic digestion and solid-phase extraction (SPE) clean-up and reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS-MS) of the resultant peptides. The clean-up step is designed to desalt the fractions and rid them of urea and ampholytes prior to analysis by LC-MS-MS. Fifty milligrams of protein were separated into 20 fractions by liquid-phase isoelectric focusing, spanning a pH range of 3-10. The effectiveness of the removal of ampholytes was monitored by capillary zone electrophoresis and LC-MS-MS. The ability to analyze all of the 20 fractions without any noticeable decrease in the separation efficiency demonstrates the overall effectiveness of the SPE clean-up step. The results show that the separation strategy is effective for high throughput characterization of proteins from complex proteomic mixtures.
Janini
George M
GM
Analytical Chemistry Laboratory, Separations Technology Group, SAIC-Frederick, Inc, National Cancer Institute at Frederick, PO Box B, Frederick, MD 21702, USA.
Conrads
Thomas P
TP
Veenstra
Timothy D
TD
Issaq
Haleem J
HJ
D004591:Electrophoresis, Polyacrylamide Gel; D010455:Peptides / Q000302:isolation & purification*; D011506:Proteins / Q000302:isolation & purification*; D020543:Proteome
D010455:Peptides; D011506:Proteins; D020543:Proteome