Pharmaceutical Sciences Faculty Publications
Two‐Dimensional Electrophoretic/Chromatographic Separations Combined with Electrospray Ionization FTICR Mass Spectrometry for High Throughput Proteome Analysis
Document Type
Article
Publication Date
2000
Journal Title
Journal of Microcolumn Separations
Volume
12
Issue
7
First Page
383
Last Page
390
DOI
10.1002/1520-667X(2000)12:7<383::AID-MCS1>3.0.CO;2-S
Abstract
A two‐dimensional separation strategy combined with electrospray ionization‐Fourier transform ion cyclotron resonance mass spectrometry (ESI‐FTICR) is being developed for high throughput proteomic analyses. Capillary isoelectric focusing (CIEF) coupled online with a robotic fraction collector is used to separate and collect microliter fractions of soluble Saccharomyces cerevisiae (yeast) proteins eluting from the capillary into a microtiter plate. Following tryptic digestion of each fraction, the resultant peptides are separated using capillary high performance liquid chromatography (HPLC) and analyzed by online ESI‐FTICR. Protein identification is based upon the use of the experimentally measured peptide masses as accurate mass tags, augmented by conventional MS/MS methods as necessary, to identify proteins predicted from the yeast genome sequence. This new separation strategy is being evaluated using proteins extracted from yeast grown in natural isotopic abundance and 15N‐enriched media. Two isotopically distinct versions of each peptide are thus observed in the ESI‐FTICR spectra. The mass differences between the two versions are used to determine the number of nitrogen atoms in the peptide, and provide an additional constraint that aids protein identification. More importantly, the use of this stable‐isotope labeling strategy enables the generation of “comparative displays” of the precise relative protein abundances. This two‐dimensional separation strategy combined with ESI‐FTICR analysis is expected to be highly amenable to automation.
Keywords
Electrophoresis, chromatography, electrospray ionization, FTICR, mass spectrometry, proteome, proteomics
Recommended Citation
Anderson, Gordon A.; Bruce, James E.; Gao, Hongying; Harkewicz, Richard; Pasa-Tolic, Ljiljana; Shen, Yufeng; Smith, Richard D.; and Veenstra, Timothy D., "Two‐Dimensional Electrophoretic/Chromatographic Separations Combined with Electrospray Ionization FTICR Mass Spectrometry for High Throughput Proteome Analysis" (2000). Pharmaceutical Sciences Faculty Publications. 506.
https://digitalcommons.cedarville.edu/pharmaceutical_sciences_publications/506